A-kinase anchoring protein-Lbc promotes pro-fibrotic signaling in cardiac fibroblasts

Biochimica et Biophysica Acta 1843 (2014) 335–345

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A-kinase anchoring protein-Lbc promotes pro-fibrotic signaling in cardiac fibroblasts Sabrina Cavin, Darko Maric, Dario Diviani ⁎ Department of Pharmacology and Toxicology, Faculté de Biologie et Médecine, University of Lausanne, 1005, Switzerland

a r t i c l e

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Article history: Received 31 May 2013 Received in revised form 30 October 2013 Accepted 13 November 2013 Available online 22 November 2013 Keywords: A kinase-anchoring protein (AKAP) Protein kinase A G protein-coupled receptor Cardiac fibroblast RhoA

a b s t r a c t In response to stress or injury the heart undergoes an adverse remodeling process associated with cardiomyocyte hypertrophy and fibrosis. Transformation of cardiac fibroblasts to myofibroblasts is a crucial event initiating the fibrotic process. Cardiac myofibroblasts invade the myocardium and secrete excess amounts of extracellular matrix proteins, which cause myocardial stiffening, cardiac dysfunctions and progression to heart failure. While several studies indicate that the small GTPase RhoA can promote profibrotic responses, the exchange factors that modulate its activity in cardiac fibroblasts are yet to be identified. In the present study, we show that AKAPLbc, an A-kinase anchoring protein (AKAP) with an intrinsic Rho-specific guanine nucleotide exchange factor (GEF) activity, is critical for activating RhoA and transducing profibrotic signals downstream of type I angiotensin II receptors (AT1Rs) in cardiac fibroblasts. In particular, our results indicate that suppression of AKAP-Lbc expression by infecting adult rat ventricular fibroblasts with lentiviruses encoding AKAP-Lbc specific short hairpin (sh) RNAs strongly reduces the ability of angiotensin II to promote RhoA activation, differentiation of cardiac fibroblasts to myofibroblasts, collagen deposition as well as myofibroblast migration. Interestingly, AT1Rs promote AKAP-Lbc activation via a pathway that requires the α subunit of the heterotrimeric G protein G12. These findings identify AKAP-Lbc as a key Rho-guanine nucleotide exchange factor modulating profibrotic responses in cardiac fibroblasts. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Cardiac fibroblasts constitute the predominant cell population in the myocardium and play a key role in maintaining the structural integrity of the heart through controlled proliferation and extracellular matrix (ECM) turnover [1,2]. In response to a neurohumoral or biomechanical stress placed on the heart, quiescent cardiac fibroblasts undergo a phenotypic transition to profibrogenic myofibroblasts, which are characterized by the expression of α-smooth muscle actin (α-SMA) [1,2], a contractile protein normally associated with smooth muscle cells. Myofibroblasts display a more mobile phenotype [3] and possess a greater synthetic ability to produce collagen-rich ECM proteins and profibrotic cytokines including the transforming growth factor β (TGF-β) [4]. Over the time, excessive formation of fibrous connective tissue and ECM leads to cardiac fibrosis. This impairs electrical coupling

Abbreviations: GTP, guanosine triphosphate; AKAP, A-kinase anchoring protein; GEF, guanine nucleotide exchange factor; AT1Rs, type I angiotensin II receptors; sh, short hairpin; ECM, extracellular matrix; α-SMA, α-smooth muscle actin; TGF-β, transforming growth factor β; Ang II, angiotensin II; MRTF, myocardin related transcription factor; α1-ARs, α1-adrenergic receptors; NVM, neonatal ventricular myocyte; AVF, adult ventricular fibroblast ⁎ Corresponding author at: Département de Pharmacologie et de Toxicologie, Rue du Bugnon 27, 1005 Lausanne, Switzerland. Tel.: +41 21 692 5404; fax: +41 21 692 5355. E-mail address: [email protected] (D. Diviani). 0167-4889/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbamcr.2013.11.008

between cardiomyocytes and increases myocardial stiffness, leading arrhythmias and severe diastolic dysfunctions. The key role of cardiac fibroblast in the cardiac remodeling process associated with cardiac hypertrophy and heart failure makes them an attractive target for the treatment of heart failure [1]. Among the factors produced in the remodeling heart that affect the phenotype and function of cardiac fibroblasts, angiotensin II (Ang II) has been shown to contribute importantly to the development of cardiac fibrosis [1,3]. In this respect, several studies indicate that the profibrotic actions of Ang II are mainly mediated by the type 1 angiotensin II receptor (AT1R), a G protein coupled receptor linked to the heterotrimeric G proteins Gq and G12/13 [5]. By promoting myofibroblast transdifferentiation, activated AT1Rs favor ECM accumulation and TGFβ-1 synthesis, and, as a consequence, fibrosis [3,6]. Several lines of evidence now suggest that the small molecular weight GTPase RhoA plays a crucial role in mediating several profibrotic responses in cardiac fibroblasts [7–10]. In this respect, recent findings indicate that RhoA can promote α-SMA expression in cardiac fibroblasts through a signaling pathway that involves actin polymerization and the nuclear translocation of myocardin related transcription factors (MRTFs) [9,10]. Moreover, exposure of cardiac myofibroblasts to an inhibitor of Rho-kinase, a downstream effector of RhoA, inhibits cardiac fibroblast migration [11]. Finally, disruption of the gene encoding the Rho-kinase ROCK1 reduces collagen deposition and prevents reactive cardiac fibrosis induced by Ang II infusion or pressure overload [12,13].

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Rho GTPases act as molecular switches, cycling between active GTPbound and inactive GDP-bound states. In vivo, the cycling between the GDP- and GTP-bound forms is regulated by guanine nucleotideexchange factors (GEFs) that stimulate the exchange of GDP with GTP. We have previously identified an exchange factor mainly expressed in the heart, termed AKAP-Lbc, which functions as GEF for RhoA (Rho-GEF) as well as an A-kinase anchoring protein (AKAP) [14]. In cardiomyocytes, the Rho-GEF activity of AKAP-Lbc Rho-GEF is enhanced in response to the activation of α1-adrenergic receptors (ARs) through a signaling pathway that requires the α subunit of the heterotrimeric G protein G12 [14,15]. Silencing AKAP-Lbc expression in rat neonatal ventricular myocytes (NVMs) strongly inhibits the ability of α1-ARs to induce RhoA activation and hypertrophic responses, suggesting a role for this anchoring protein in cardiac remodeling [15,16]. While the implication of RhoA signaling in cardiac fibrosis has been proposed by several studies, the identity of the Rho-GEFs that transduces fibrotic signals in cardiac fibroblasts has remained elusive. In the present study, we used a lentivirus-based strategy to deliver AKAP-Lbc-specific short hairpin (sh) RNAs into primary cultures of rat adult ventricular fibroblasts (AVFs) or myofibroblasts (AVMyoFs). Using this approach, we could demonstrate that AKAP-Lbc plays a key role in mediating Ang II-induced fibrotic responses. In particular, we found that AKAP-Lbc participates in a signaling cascade activated by AT1Rs that includes Gα12, AKAP-Lbc, and RhoA. This pathway promotes α-SMA expression and myofibroblast differentiation and associated increased TGF-β1 production, collagen deposition as well as myofibroblast motility. Collectively, these findings identify AKAP-Lbc as the first RhoGEF regulating profibrotic signals in cardiac fibroblasts.

2. Material and methods 2.1. Expression constructs Double-stranded hairpin oligonucleotides based upon rat AKAP-Lbc mRNA sequences (GI: 198386327, bases 6347–6365 and 6626–6644) were cloned into the HindIII and BglII sites in the pSUPER vector. The following oligonucleotide sequences were used: AKAP-Lbc shRNA1 (sense strand) 5′-GCAAGTCGATCATGAGAAT-3′; AKAP-Lbc shRNA2 (sense strand) 5′-GGATAAGCGTTTCCAAGCC-3′; mutated AKAP-Lbc shRNA (sense strand) 5′-GCATGTCGATCATGCGATT-3′. Underlined base pairs in the mutated shRNA differ from the wild-type AKAP-Lbc shRNA. To generate lentiviral transfer vectors encoding AKAP-Lbc shRNAs, cDNA fragments containing the H1 RNA polymerase III promoter as well as the sequences encoding shRNAs were excised using BamHI/SalI from the pSUPER vector and subcloned into the pAB286.1 transfer vector. To generate the Gα12 G228A-pAB286.1 vector, a BamHI/XhoI fragment containing the CMV promoter and the entire open reading frame for the Gα12 G228A mutant was PCR-amplified from the Gα12 G228A-pCDNA3.1 vector and subcloned into the BamHI and SalI sites in the pAB286.1 vector. To generate the RhoA N19pAB286.1 vector, a BamHI/SalI fragment containing the CMV promoter and the entire open reading frame for the RhoA N19 mutant was PCRamplified from the RhoA N19-pFLAG-CMV-6c vector and subcloned into the BamHI and SalI sites in the pAB286.1 vector. The lentiviral packaging vectors pCMVDR8.91 and pMD2.VSVG encode the viral capsid and the vesicular stomatitis virus-G envelope protein, respectively. Plasmids encoding GFP fusions of AKAP-Lbc and AKAP-Lbc Y2153F were described previously [14].

2.2. Peptide synthesis Arg11-SuperAKAP-IS (Arg11-QIEYVAKQIVDYAIHQA) and Arg11scrambled SuperAKAP-IS (Arg11-QDVEIHVKAAYYQQIAI) were synthesized and purified to N 90% purity (GenWay Biotech).

2.3. Preparation of adult rat ventricular fibroblasts AVFs were isolated from 10-week-old male Sprague–Dawley rats. Briefly animals were euthanized in a prefilled CO2 chamber with 100% concentration of CO2, and excised hearts were washed in ice-cold 1× ADS buffer (116 mM NaCl, 20 mM HEPES, 1 mM NaH2PO4, 5.5 mM D-glucose, 5.4 mM KCl, 0.8 mM MgSO4). Ventricular tissues were minced into small pieces and incubated overnight in a solution of 0.5 mg/ml trypsin (USB) at 4 °C on an orbital shaker at 80 rpm. Trypsinized tissues subsequently underwent four cycles of enzymatic digestion for 7 min at 37 °C in a solution of 80 mg/ml of type II collagenase (Worthington). Cells were pelleted by centrifugation (800 rpm, 10 min) and resuspended in DMEM supplemented with 10% FBS and penicillin/streptomycin (Gibco). The resuspended cells were then passed through a 70 μm-cell strainer, seeded onto uncoated plastic dishes and incubated for 2 h at 37 °C with 10% CO2. DMEM was subsequently replaced with EGM-2 to maintain cardiac fibroblasts in an undifferentiated state [17]. Differentiation of AVFs to AVMyoFs was induced by culturing cells in DMEM supplemented with 10% FBS and penicillin/streptomycin. Purity of AVFs and AVMyoFs preparations was determined by immunofluorescence staining using mouse monoclonal anti-α-actinin (Sigma, 1:500 dilution), rabbit polyclonal anti-desmin (Cell Signaling, 1:25 dilution) and mouse monoclonal cy3-conjugated anti-vimentin antibodies (Sigma, dilution = 1:250). The presence of contaminant endothelial cells was assessed by incubating primary cultures for 4 h in the presence of Dil-acyl LDL (Biomedical Technologies Inc., dilution = 1:20). Cultures contained almost exclusively primary ventricular fibroblasts, as N95% of the cells were positive for the fibroblast marker vimentin, were negative for the cardiomyocyte marker α-actinin and the vascular smooth-muscle cell marker desmin, and were not labeled with Dil-acyl LDL (see Fig. S1A–D, in Supplementary material). Neonatal rat cardiomyocytes were used as a positive control for α-actinin and desmin staining and as a negative control for vimentin staining. The endothelial EA.hy 926 cell line was used as a positive control for Dil-acyl LDL labeling. Western blot analysis of AVF lysates confirmed the presence of vimentin but not α-actinin and desmin (see Fig. S1E–G, in Supplementary material). AVMyoF transdifferentiation was assessed by staining cells using mouse monoclonal anti-α-SMA antibodies (Sigma, dilution = 1:500) (see Fig. S1H, in Supplementary material). 2.4. Cell culture and treatment Rat AVFs or AVMyoFs were seeded on 6-cm uncoated plastic dishes at 6 × 105 cells per dish for Rhotekin pulldown experiments, Western blots and real time PCR experiments. For indirect immunofluorescence experiments, ventricular fibroblasts were seeded on 4.2 cm2 TPX LabTek chamber slides at 5 × 104 cells per chamber. For [3H]-proline incorporation assays, AVFs were seeded on uncoated 25-mm wells at 3 × 105 cells per well. When indicated, cells were treated with losartan (1 μM; Sigma), SB203580 (10 μM; Calbiochem), PD123319 (1 μM; Sigma), BMS-345541 (5 μM; Sigma), RO318220 (10 μM; Sigma), Gö6976 (0.5 μM; Sigma), or Na3V04 (1 mM; Sigma) 6 h prior stimulation with Ang II (100 nM; Sigma). 2.5. Production of lentiviruses VSV-G pseudotyped lentiviruses were produced by cotransfecting 293-T cells with 20 μg of the pAB286.1 vectors encoding either the AKAP-Lbc shRNAs, the Gα12 G228A dominant negative mutant, or the Flag-RhoA N19 construct, 15 μg of pCMVDR8.91, and 5 μg of pMD2.VSVG using the calcium phosphate method. Culture medium was replaced by serum-free DMEM at 12 h after transfection. Cell supernatants were collected 48 h later, filtered through a 0.22-μm filter unit and concentrated using Centricon-Plus-70 MW 100,000 columns (Millipore). Virus titers were determined by infecting HEK293 cells using serial dilutions of the viral stocks and by scoring puromycin-

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resistant clones (at 6 days after infection). Titers determined using this method were between 1 × 108 and 8 × 108 TU/ml. 2.6. Lentiviral infection AVFs or AVMyoFs were infected 24 h after plating using pAB286.1based lentiviruses encoding wild type or mutated AKAP-Lbc shRNAs, the Gα12 G228A mutant or the Flag-RhoA N19 construct at a multiplicity of infection (MOI) of 50 in EGM-2 or DMEM supplemented with 10% FBS, containing 8 μg/ml of polybrene. 48 h after infection, culture media were replaced and fibroblasts incubated for five additional days in EGM-2 or DMEM supplemented with 10% FBS. For rescue experiments, AVFs were transfected 4 days after infection with the cDNA encoding human AKAP-Lbc-GFP or the AKAP-Lbc Y2153F-GFP mutant using Lipofectamine 2000. Transfection was performed in EGM-2 or DMEM in the absence of antibiotics for a period of 6 h. 20 h after transfection, cells were serum starved for 24 h and stimulated with 10−7 M Ang II for an additional 24 h. Pools of 104 GFP positive (i.e. expressing AKAP-Lbc-GFP constructs) or GFP negative (i.e. non-transfected) cells were sorted by FACS and used for quantitative PCR experiments (see below). 2.7. Expression and purification of recombinant proteins in bacteria GST-tagged fusion proteins of the Rho-binding domain (RBD) of Rhotekin were expressed using the bacterial expression vector pGEX4T1 in the Bl21DE3 strain of Escherichia coli and purified. To induce the expression of fusion proteins, exponentially growing bacterial cultures were incubated for 24 h at 16 °C with 1 mM isopropyl 1-thio-βD-galactopyranoside (IPTG), and subsequently subjected to centrifugation. Pelleted bacteria were lysed in buffer A (50 mM Tris, pH 7.4, 500 mM NaCl, 10 mM MgCl2, 1% (w/v) Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 4 μg/ml leupeptin, 2 μg/ml aprotinin, 2 μg/ml pepstatin, 0.1 mM phenylmethylsulfonyl fluoride), sonicated, and centrifuged at 34,000 ×g for 30 min at 4 °C. After incubating the supernatants with glutathione sepharose beads (Amersham Biosciences) for 1 h at 4 °C, the resin was washed five times with 10 volumes of buffer A. The protein content of the beads was assessed by Coomassie Blue staining of SDS-polyacrylamide gels. Beads were used immediately for the Rhotekin RBD pulldown assay.

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leupeptin and 1 mM PMSF). 2 mg of dialysed lysates were then incubated for 4 h at 4 °C with 4 μg of rabbit polyclonal anti-AKAP-Lbc antibodies (Covance) and 20 μl of Protein-A-sepharose beads to immunoprecipitate endogenous AKAP-Lbc proteins. Following a brief centrifugation on a bench-top centrifuge, the pelleted beads were washed five times with buffer C and twice with PBS and proteins were eluted in SDS-PAGE sample buffer by boiling samples for 3 min at 95 °C. For immunoprecipitation of Flag-RhoA N19, rat AVFs infected with lentiviruses encoding for the Flag-RhoA N19 mutant were lysed 7 days after infection in 1 ml of buffer D (20 mM Tris, pH 7.4, 150 mM NaCl, 1% (w/v) Triton X-100, 4 μg/ml leupeptin, 2 μg/ml aprotinin, 2 μg/ml pepstatin, 0.1 mM phenylmethylsulfonyl fluoride). Cell lysates were incubated for 1 h at 4 °C on a rotating wheel. The solubilized material was centrifuged at 34,000 ×g for 30 min at 4 °C, and the supernatants were incubated for 4 h at 4 °C with 20 μl of anti-Flag M2 affinity resin (Sigma). Following a brief centrifugation on a bench-top centrifuge, the pelleted beads were washed five times with buffer D and proteins were eluted in SDS-PAGE sample buffer by boiling samples for 5 min at 95 °C. Eluted proteins were analyzed by SDS-PAGE and Western blotting. 2.10. SDS-PAGE and Western blotting Samples denatured in SDS-PAGE sample buffer were separated on acrylamide gels and electroblotted onto nitrocellulose membranes. The blots were incubated with primary antibodies and horseradishconjugated secondary antibodies (Amersham Biosciences) as previously indicated [29]. The following affinity-purified primary antibodies were used for immunoblotting: mouse monoclonal anti-RhoA (Santa Cruz, 1:250 dilution), mouse monoclonal anti-α-SMA (Sigma, 1:2000 dilution), rabbit polyclonal anti-Gα12 (Santa Cruz, 1:250 dilution), mouse monoclonal anti-actin (Sigma, 1:2000 dilution), mouse monoclonal anti-Flag (Sigma, 1:2000 dilution), mouse monoclonal anti-α-actinin (Sigma, 1:500 dilution), rabbit polyclonal anti-desmin (Cell Signaling, 1:1000 dilution), and mouse monoclonal antivimentin (Santa Cruz, 1:200). Antibodies to AKAP-Lbc were generated in rabbits using a KLH conjugated peptide (residues 2671–2694) as the immunogen (Covance). AKAP-Lbc serum was used at a 1:2000 dilution for immunoblots.

2.8. Rhotekin Rho-binding domain (RBD) pulldown assay

2.11. RNA extraction and real-time RT-PCR

Infected rat AVFs or AVMyoFs grown for 6 days in EGM-2 medium or in DMEM supplemented with 10% FBS were starved for 24 h in the absence of serum. Cells were then incubated for 15 min with or without 10−7 M Ang II and lysed in RBD lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 30 mM MgCl2, 1 mM dithiothreitol, 0.5% (w/v) Triton X-100, 4 μg/ml leupeptin, 2 μg/ml aprotinin, 2 μg/ml pepstatin, 0.1 mM phenylmethylsulfonyl fluoride). Lysates were subjected to centrifugation at 20,600 ×g for 10 min at 4 °C and incubated with 30 μg of RDB beads for 1 h at 4 °C. Beads were then washed three times with RBD buffer, resuspended in SDS-PAGE sample buffer (100 mM Tris, pH6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol), and analyzed by SDS-PAGE.

Total mRNA was extracted from rat AVFs or AVMyoFs using the RNeasy Fibrous Tissue Mini Kit (Qiagen) and single-strand cDNA was synthesized from 0.05 μg to 0.5 μg of total RNA using random hexamers (Applied Biosystems) and SuperScript II reverse transcriptase (Invitrogen). Real-time PCR was performed using the Applied Biosystems 7500 Fast Real-Time PCR System with TGF-beta1 (Rn01475963_m1), α-SMA (Rn01759928_g1), and AKAP13 (Rn01478247_m1) Taqman Gene expression Assays. β2-Microglobulin (Rn00560865_m1) mRNA was used as invariant internal control.

2.9. Immunoprecipitation experiments For immunoprecipitation of endogenous AKAP-Lbc, rat AVFs grown in 100 mm dishes were lysed in 1 ml of buffer B (20 mM Tris pH7.4, 150 mM NaCl, 1% (w/v) Triton-X-100, 0.1% sodium deoxycholate, 5 μg/ml aprotinin, 10 μg/ml leupeptin and 1 mM PMSF). Cell lysates were incubated for 4 h at 4 °C on a rotating wheel. The solubilized material was centrifuged at 34,000 ×g for 30 min at 4 °C and the supernatants were dialysed overnight against buffer C (20 mM Tris pH 7.4, 150 mM NaCl, 1% (w/v) Triton-X-100, 5 μg/ml aprotinin, 10 μg/ml

2.12. Immunocytochemistry Infected AVFs and AVMyoFs were grown for 5 days in EGM-2 medium, serum starved for an additional 24 h and finally incubated for 24 h with or without 10−7 M Ang-II. Cells were then washed twice with PBS, fixed for 10 min in PBS/3.7% formaldehyde, permeabilized for 5 min with 0.5% (w/v) Triton X-100 in PBS, and blocked for 1 h in PBS/ 1%BSA. The expression of α-SMA was assessed by incubating fibroblasts for 1 h with 1:500 dilution of the mouse anti-α-SMA monoclonal antibody (Sigma) followed by 1 h incubation with 1:250 dilution of Alexa Fluor 488-conjugated anti-mouse secondary antibodies (Molecular Probes). Immunofluorescent staining was visualized using a Zeiss Axiophot fluorescence microscope.

S. Cavin et al. / Biochimica et Biophysica Acta 1843 (2014) 335–345

2.14. Invasion and migration assays Invasion assays were performed using a modified Boyden chamber technique that measures the ability of adult rat ventricular fibroblasts to migrate across a matrix barrier in response to Ang II. We used 24-well tissue culture plates with inserts containing an 8 μm pore-size polycarbonate membrane coated with Matrigel basement membrane matrix (BD Biosciences). Rat AVMyoFs were quiesced in DMEM containing 0.6% FCS for 24 h. Cells were then trypsinized, resuspended in DMEM containing 0.6% FCS and counted. 5 × 104 cells were loaded into the upper chamber. The lower chamber was filled with DMEM supplemented with 0.6% FCS and 10−7 M Ang II. After a 24-h incubation period, the cell suspension was aspirated, and the membranes were fixed in 70% ethanol at −20 °C for 30 min. Cells that had attached but not migrated were removed, the membranes were rinsed in water, and migrated cells on the underside of the membrane were visualized with staining in 2% crystal violet. Cells were counted in 10 random fields with a light microscope. Migration assays were performed as for invasion assays, except that 104 cells were loaded on the top of uncoated membranes (i.e. no Matrigel) and that the incubation period in the presence of Ang II was of 8 h. 2.15. Statistical analysis Data are presented as means ± SE. All statistical analyses were performed using one-way ANOVA with Tukey's multiple comparison tests. 3. Results

140 120 100 80 60 40 20 0

kDa

AKAP-Lbc 250 NVMs

1

AVFs

2

Mutated Control shRNA 1 shRNA 2 shRNA

C) kDa 1)

AVMyoFs

B) AVFs

Infected AVFs were grown for 5 days in EGM-2 medium and for an additional 24 h in the absence of serum. Cells were then incubated for 24 h with ascorbic acid (50 μg/ml), [3H]-proline (5 μCi/ml) and with or without 10−7 M Ang II. Collagen secretion was then measured as previously described [18].

A) Relative AKAP-Lbc mRNA expression (%)

2.13. [3H]-proline incorporation assay

- Ang II - Ang II - Ang II -

Ang II

25

Rho-GTP

20 2)

25 20

3)

250

Total RhoA AKAP-Lbc

50 Actin

4) 37 1

2

3

4

5

6

7

8

D) Rho-GTP (% of control)

338

1200

Untreated Ang II

1000 800 600 400

*

*

200 0

3.1. AKAP-Lbc is involved in angiotensin II-mediated RhoA activation in rat AVFs Among the profibrotic factors that are secreted in the myocardium in response to stress, Ang II has been shown to play a major role in mediating the cellular responses that ultimately lead to cardiac fibrosis [19–22]. While the implication of Rho GTPases in the pro-fibrotic pathways activated by Ang II is well established [12], the identity of the RhoGEFs mediating these pathological responses in cardiac fibroblasts has remained elusive. In this context, we recently found that the RhoAspecific GEF AKAP-Lbc is highly enriched in cardiac tissues where it plays a key role in modulating the response of cardiomyocytes to stress [14,15]. We now show, both by real time quantitative PCR and by Western blot, that AKAP-Lbc is expressed in primary cultures of rat AVFs and AVMyoFs (Fig. 1A and B) thus raising the possibility that it might participate in the transduction of signals that initiate cardiac fibrosis. To address this question, we initially assessed the impact of inhibiting AKAP-Lbc expression by RNA interference on the ability of Ang II to induce RhoA activation in rat AVFs. Short hairpin (sh) RNAs specific to AKAP-Lbc or control shRNAs were delivered into rat AVFs using lentiviral vectors. Gene silencing was confirmed upon immunoblot analysis of cell lysates using AKAP-Lbc specific antibodies (Fig. 1C, panel 3). RhoA activation was assessed using the Rhotekin pulldown assay on cells treated for 15 min with 10−7 M Ang II based on the observation that RhoA activation peaked at this time point (see Fig. S2, upper panel, in Supplementary material). Interestingly, infection of rat AVFs with lentiviruses encoding AKAP-Lbc specific shRNAs caused a near complete inhibition of the ability of Ang II to induce the formation of

Control shRNA 1 shRNA 2 Mutated shRNA Fig. 1. AKAP-Lbc mediates Ang II-induced activation of RhoA in adult ventricular fibroblasts. A) Real time quantitative PCR analysis of AKAP-Lbc expression in rat NVMs and AVFs. B) Western blot analysis of AKAP-Lbc expression in lysates from rat AVFs and AVMyoFs. C) Rat AVFs were infected with control lentiviruses or with lentiviruses encoding wild type or mutated AKAP-Lbc shRNAs at a MOI of 50. Six days after infection cells were incubated for 15 min with or without 10−7 M Ang II. Cell lysates were incubated with GST-RBD beads. The bound RhoA was detected using a monoclonal anti-RhoA antibody (panel 1). The relative amounts of total RhoA (panel 2), AKAP-Lbc (panel 3) and actin (panel 4) in the cell lysates were detected using specific antibodies, as indicated. D) Quantitative analysis of the GTP-RhoA associated with RBD beads was obtained by densitometry. The amount of RhoA bound to RBD (panel 1) was normalized to the RhoA content of cell extracts (panel 2). Results are expressed as mean ± S.E. of 3 independent experiments. *P b 0.05 as compared with Rho-GTP levels measured in Ang II-stimulated rat AVFs infected with mutated shRNAs.

active GTP-bound RhoA as compared to cells infected with control lentiviruses or lentiviruses expressing the mutated AKAP-Lbc shRNA (Fig. 1C, panel 1, and D). This suggests that AKAP-Lbc plays a central role in mediating the effects of Ang II on RhoA activation in ventricular fibroblasts. Evidence accumulated over the last few years indicates that AKAPLbc can anchor multiple signaling enzymes including kinases such as PKA, PKC, PKD, MAP kinases and IKKβ, and tyrosine phosphatases such as Shp2 that could potentially modulate RhoA signaling [14,16,23–27]. This raises the possibility that the inhibitory effect of AKAP-Lbc silencing on Ang-II-mediated RhoA activation (Fig. 1C) could reflect the unhooking of these transduction enzymes from the RhoA pathway.

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Based on our previous findings showing that AKAP-Lbc is a Gα12activated Rho-GEF [14], we then decided to assess the contribution of Gα12 in mediating the activation of the AKAP-Lbc–RhoA pathway downstream the AT1 receptors. To address this issue, rat AVFs were infected with lentiviruses encoding a dominant negative mutant of Gα12 (Gα12 G228A) prior to stimulation with 10−7 M Ang II. We observed that overexpression of the Gα12 G228A mutant inhibits by 77% the activation of RhoA induced by Ang II (Fig. 2A and B), suggesting that, in rat AVFs, AT1 receptors mediate RhoA activation via Gα12. In order to provide direct evidence that Ang II induces AKAP-Lbc activation via AT1 receptors and Gα12, we determined whether losartan treatment and expression of the Gα12 G228A mutant could impair the interaction between endogenous AKAP-Lbc and RhoA in rat AVFs. Importantly, we have previously shown that the formation of AKAP-Lbc– RhoA complexes reflects the activation state of AKAP-Lbc [32]. Rat AVFs were incubated with 1 μM losartan (Fig. 2C and D) or infected with lentiviruses encoding for the Gα12 G228A mutant (Fig. 2E and F) prior to stimulation with 10− 7 M Ang II. Endogenous AKAP-Lbc was then immunoprecipitated using anti-AKAP-Lbc antibodies and the presence of associated RhoA determined by immunoblot. Stimulation of cells with Ang II induced a significant increase in the amount of RhoA associated with AKAP-Lbc (Fig. 2C and E, upper panel, lane 2). This effect was completely abolished by losartan treatment (Fig. 2C, upper panel, lane 4; and D) and significantly impaired following the expression of the Gα12 G228A mutant (Fig. 2E, panel 1, lane 4; and F), indicating that Ang II induces AKAP-Lbc activation via AT1 receptors and Gα12. Collectively, these findings strongly suggest that, in adult ventricular fibroblasts, Gα12 and AKAP-Lbc control RhoA activation downstream of AT1 receptors.

To address this question we initially determined whether inhibition of type II PKA anchoring in adult fibroblasts affected RhoA activity. Treatment of rat AVFs with cell permeable type II PKA anchoring inhibitory peptides (SuperAKAP-IS) or control scrambled peptides (scrambled SuperAKAP-IS) [28] did not influence the formation of RhoA-GTP induced by Ang II (see Fig. S3A, upper panel, lanes 2 and 4, and B, in Supplementary material). However, in line with previous evidence indicating that anchored PKA inhibits the basal Rho-GEF activity of AKAP-Lbc [29,30], inhibition of type II PKA anchoring significantly enhanced basal RhoA activation (see Fig. S3A, upper panel, lanes 1 and 3, and B). Additional control experiments revealed that inhibition of p38, MEK1, PKC, PKD, IKKβ, and tyrosine phosphatases using the inhibitors, SB203580 and PD098059, RO318220, Gö6976, BMS-345541 and sodium orthovanadate (Na3VO4), respectively, did not affect in a significant manner Ang II-mediated RhoA-GTP formation (see Fig. S3C, upper panel, and D, in Supplementary material). These findings indicate that the kinases and phosphatases anchored by AKAP-Lbc do not modulate the effects of Ang II on RhoA signaling in rat AVFs. Based on these findings, we undertook experiments aimed at determining the molecular players linking Ang II to the activation of AKAPLbc–RhoA signaling pathway in AVFs. Ventricular fibroblasts express two different receptors for Ang II, namely AT1 receptors and AT2 receptors [31]. To assess which of these receptor subtypes promotes RhoA activation, rat AVFs were incubated with 1 μM losartan, an AT1 receptors antagonist, or 1 μM PD123319, an AT2 receptors antagonist, prior Ang II stimulation. RhoA-GTP formation was totally blocked by pre-treatment with losartan but was not with PD123319 (see Fig. S4A, upper panel, and B, in Supplementary material), suggesting that AT1 receptors are the main receptor subtype mediating RhoA activation in rat AVFs.

A Control

- AngII - AngII 20

2)

- AngII - AngII

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25 20

IP: anti-AKAP-Lbc IB: anti-RhoA

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25

Cell extracts IB: anti-RhoA

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20

G 12 G228A

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37

12

250

IP: anti-AKAP-Lbc IB: anti-AKAP-Lbc

3)

2

3

4

1

D Untreated Ang II

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G 12 G228A

Immunoprecipitated RhoA (% of control)

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120 100

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3

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80 60

40 20 0

* Control Losartan

Cell extracts IB: anti-G 12

4)

250 1

600 500 400 300 200 100 0

IP: anti-AKAP-Lbc IB: anti-RhoA

25 Total RhoA

kDa

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1)

Rho-GTP

25 20

Control

Control Losartan kDa

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Rho-GTP (% of control)

E

37

F Immunopprecipitated RhoA(%of control)

kDa

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G 12 G228A

339

1

2

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Fig. 2. Gα12 mediates Ang II-induced AKAP-Lbc activation in adult ventricular fibroblasts. A) Rat AVFs were infected with control lentiviruses or lentiviruses encoding the Gα12 G228A mutant at a MOI of 50. Six days after infection cells were incubated for 15 min with or without 10−7 M Ang II. GST-RBD pulldown assays were performed as described in Fig. 1C. The bound RhoA (top) and the relative amounts of total RhoA (middle) and Gα12 G228A (bottom) in the cell lysates were assessed by using specific antibodies, as indicated. B) Quantitative analysis of the GTP-RhoA associated with RBD beads was obtained by densitometry as indicated in Fig. 1D. Results are expressed as mean ± S.E. of 7 independent experiments. *P b 0.05 as compared with Rho-GTP levels measured in Ang II-stimulated AVFs infected with control lentiviruses. C) Rat AVFs pre-treated or not with 1 μM losartan 6 h prior to incubation with or without Ang II at 10−7 M for 15 min. Cell lysates were subjected to immunoprecipitation (IP) using anti-AKAP-Lbc antibodies. Western blots of the immunoprecipitates and the cell extracts were revealed using antibodies against RhoA (panels 1 and 2) and AKAP-Lbc (panel 3). D) Quantitative analysis of the amount of RhoA immunoprecipitated with AKAP-Lbc was obtained by densitometry. Results are expressed as mean ± S.E. of 3 different experiments. *P b 0.05 as compared with levels of co-immunoprecipitated RhoA measured in Ang IItreated control rat AVFs. E) Rat AVFs were infected with control lentiviruses or lentiviruses encoding the Gα12 G228A mutant at a MOI of 50. Six days after infection, cells were incubated for 24 h with or without 10−7 M Ang II and cell lysates were subjected to immunoprecipitation (IP) using anti-AKAP-Lbc antibodies. Western blots of the immunoprecipitates and the cell extracts were revealed using specific antibodies, as indicated. F) Quantitative analysis of the amount of RhoA immunoprecipitated with AKAP-Lbc was obtained by densitometry. Results are expressed as mean ± S.E. of 4 different experiments. *P b 0.05 as compared with levels of co-immunoprecipitated RhoA measured in Ang II-treated rat AVFs infected with control lentiviruses.

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3.2. AKAP-Lbc controls α-SMA expression and associated phenotypic conversion of cardiac fibroblasts to myofibroblasts

AKAP-Lbc shRNAs, and subsequently transfected with the plasmids encoding GFP fusion proteins of AKAP-Lbc or the AKAP-Lbc Y2153F mutant that lacks Rho GEF activity [14]. GFP negative (i.e. silenced cells) and GFP positive cells (i.e. cells rescued using AKAP-Lbc GFP constructs) were sorted by FACS and α-SMA expression determined by real-time PCR. Our results indicate that rescue with wild type AKAP-Lbc fully restored Ang II-mediated transcription of α-SMA, whereas rescue with the AKAP-Lbc Y2153F mutant did not (Fig. 3D). Collectively, these findings indicate that the AKAP-Lbc–RhoA pathway plays a crucial role in mediating the effects of Ang II on cardiac myofibroblast differentiation. A similar inhibition of Ang II-induced α-SMA expression was observed following incubation of rat AVFs with the AT1R inhibitor losartan (see Fig. S5A, upper panel lane 4, in Supplementary material) but not with AT2R inhibitor PD123319 (see Fig. S5A, upper panel, lane 6, in Supplementary material). Importantly, the effects of AT1Rs on cardiac myofibroblast differentiation were shown to require Gα12 and RhoA since infection of rat AVFs with lentiviruses expressing dominant negative mutants of Gα12 (G228A) or RhoA (Flag-RhoA N19) significantly decreased Ang-II-induced α-SMA expression (see Fig. S5B, upper panel, lanes 4 and 6, in Supplementary material). To confirm the implication of AKAP-Lbc in the process of cardiac myofibroblast formation induced by Ang II, we determined the impact of AKAP-Lbc silencing on the ability of Ang II to promote morphological changes and cytoskeletal reorganization in rat AVFs by immunofluorescence microscopy. Treatment of rat AVFs infected with control lentiviruses with Ang II for 24 h induced the assembly of α-SMA positive stress fiber characteristic of myofibroblasts (Fig. 4A and B) in about 54% to 58% of the cells. Ang II treated cells were also larger with a

Myofibroblast formation is a process crucially involved in the pathology of cardiac fibrosis. Recent evidence indicates that RhoA can regulate myofibroblast differentiation by promoting the nuclear translocation of the serum response factor (SRF) and myocardin related transcription factors (MRTFs), which, in turn, enhance transcription and expression of smooth muscle specific genes such as α-SMA [8,10]. Based on these findings and our current results identifying AKAP-Lbc as the mediator of Ang II-induced RhoA activation in ventricular fibroblasts, we investigated the possibility that this anchoring protein could control α-SMA expression and differentiation of cardiac fibroblasts to myofibroblasts. To address this question, we initially determined whether silencing the expression of AKAP-Lbc could affect Ang II-mediated induction of the myofibroblast marker α-SMA. Treatment of rat AVFs infected with control lentiviruses or lentiviruses encoding mutated AKAP-Lbc shRNAs with 10−7 M of Ang II for 24 h induced a 2.4 to 2.6 fold increase in α-SMA expression as assessed by immunoblot (Fig. 3A, upper panel, lanes 2 and 8, and B) and by real-time PCR (Fig. 3C). Interestingly, the stimulatory effect of Ang II on the expression of α-SMA mRNAs and proteins was impaired by more than 90% in rat AVFs expressing AKAP-Lbc shRNAs (Fig. 3A, lanes 4 and 6, B and C). To provide a formal demonstration that the effects of AKAP-Lbc and α-SMA expression are mediated by RhoA we performed experiments that combined silencing of the endogenous anchoring protein and rescue with human AKAP-Lbc forms that are refractory to the rat-specific shRNA (Fig. 3D). Rat AVFs were infected with lentiviruses encoding

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Fig. 3. AKAP-Lbc mediates Ang II-induced α-SMA expression in AVFs. A) Rat AVFs were infected with control lentiviruses or with lentiviruses encoding wild type or mutated AKAP-Lbc shRNAs at a MOI of 50. Six days after infection, cells were incubated for 24 h in the absence or presence of 10−7 M Ang II. The relative expression of α-SMA and AKAP-Lbc in the cell lysates was assessed by using a monoclonal antibody against α-SMA (upper panel) and a polyclonal antibody against AKAP-Lbc (middle panel), respectively. Total proteins were resolved by SDSPAGE and Ponceau S staining (lower panel). B) Quantitative analysis of α-SMA expression was obtained by densitometry. The amount of α-SMA was normalized to the total amount of proteins. Results are expressed as mean ± S.E. of 3 independent experiments. *P b 0.05 as compared with α-SMA levels measured in Ang II-treated rat AVFs infected with lentiviruses encoding mutated AKAP-Lbc shRNAs. C) Rat AVFs were infected and treated as indicated in A. Total RNA samples were extracted and a real-time PCR analysis of α-SMA mRNA expression was performed. All results are expressed as mean ± S.E. of 4 independent experiments. *P b 0.05 as compared with mRNA levels measured in Ang II-stimulated rat AVFs infected with mutated shRNAs. D) Real-time PCR analysis of α-SMA expression was performed on total RNA preparations obtained from FACS-sorted rat AVFs that were infected with lentiviruses encoding AKAP-Lbc shRNAs as indicated in A and subsequently transfected with rescue constructs encoding GFP-tagged AKAP-Lbc or AKAP-Lbc Y2153F. Results are expressed as mean ± S.E. of 3 independent experiments. *P b 0.05.

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Fig. 4. AKAP-Lbc mediates Ang II-induced ventricular myofibroblast differentiation. A) Rat AVFs were infected and treated as indicated in Fig. 3A. Cells were then fixed, permeabilized and incubated with anti-α-SMA monoclonal antibodies as well as Alexa Fluor 488-conjugated anti-mouse secondary antibodies to detect the expression of α-SMA. B) Percentage of myofibroblasts measured on 20 random fields per condition. C) Western blot analysis of the expression of AKAP-Lbc (upper panel) and actin (bottom panel) performed on protein extracts from rat AVFs analyzed in A.

more rigid appearance. AKAP-Lbc silencing abolished Ang II-induced α-SMA upregulation as well as morphological changes and cytoskeletal reorganization (Figs. 4A and 2B). Silencing efficiency was evaluated by immunoblot using anti-AKAP-Lbc antibodies (Fig. 4C). Taken together, these results suggest that the Gα12–AKAP-Lbc–RhoA pathway controls α-SMA expression and the phenotypic transition of cardiac fibroblasts to myofibroblasts. 3.3. AKAP-Lbc mediates Ang II-induced collagen synthesis in rat AVFs Based on the above results and knowing that cardiac myofibroblasts produce and secrete excessive amounts of collagen-rich extracellular matrix that contribute to cardiac fibrosis and dysfunction [1], we determined the impact of AKAP-Lbc silencing on the ability of Ang II to promote ECM production in rat AVFs by assaying collagenase-sensitive [3H]-proline incorporation. Incubation of rat AVFs infected with control lentiviruses or lentiviruses encoding mutated AKAP-Lbc shRNAs with 10−7 M Ang II increased collagenase-sensitive [3H]-proline incorporation by 2.1 to 2.3 fold compared to untreated cells (Fig. 5A). This effect was impaired by about 90% following the suppression of AKAP-Lbc expression (Fig. 5A). AKAP-Lbc silencing expression was confirmed by immunoblot (Fig. 5B). These findings support the idea that AKAP-Lbc mediates the upregulation of collagen production in cardiac fibroblasts in response to Ang II stimulation. 3.4. AKAP-Lbc mediates Ang II-induced TGF-β1 expression in rat AVFs Strong evidence supports the notion that Ang II, through the stimulation of AT1Rs, enhances the secretion of profibrotic ligands such as

TGF-β from activated cardiac fibroblasts [1,6]. This favors the amplification of the fibrotic response. Based on this observation, we determined whether inhibition of AKAP-Lbc expression in rat AVFs could affect Ang II-induced TGF-β1 expression as assessed by real time quantitative PCR. We observed that Ang II treatment of control AVFs for 6 h with 10−7 M Ang II increases TGF-β1 mRNA expression by around 2.1 fold (Fig. 6A). Infection of rat AVFs with lentiviruses encoding AKAP-Lbc shRNAs totally inhibited the ability of 10−7 M Ang II to induce TGF-β1 expression as compared to cells infected with control lentiviruses or lentiviruses encoding mutated AKAP-Lbc shRNAs (Fig. 6A). Importantly, rescue with wild type AKAP-Lbc fully restored Ang II-mediated transcription of α-SMA in silenced cells, whereas rescue with the AKAP-Lbc Y2153F mutant did not (Fig. 6B). Collectively, these results further confirm the implication of the AKAP-Lbc–RhoA pathway in the fibrotic response induced by Ang II in primary cardiac fibroblasts.

3.5. Silencing of AKAP-Lbc expression impairs Ang II-induced myofibroblast migration and invasion Differentiated cardiac myofibroblasts display increased migratory properties, which enhance their ability to invade the myocardium. Previous studies suggested that myofibroblast migration relies on the activity of Rho-GTPases, which, by regulating remodeling of the cell cytoskeleton, control cell movement [11]. Our findings showing that AKAP-Lbc mediates Ang II-induced RhoA activation in both rat AVFs (Fig. 1C and D) and AVMyoFs (see Fig. S6A, upper panel, lanes 4 and 6, in Supplementary material) prompted us to investigate the possible implication of this anchoring protein in cardiac myofibroblast migration.

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37 Fig. 5. AKAP-Lbc mediates Ang II-induced collagen production in ventricular fibroblasts. A) Rat AVFs were infected as indicated in Fig. 3. Six days after infection, cells were incubated for 24 h with 5 μCi/ml of 3H-proline in the absence or presence of Ang II at 10−7 M. Collagen synthesis was assessed by measuring 3H-proline incorporation into collagenasesensitive proteins. Results are expressed as mean ± S.E. of 4 independent experiments. *P b 0.05 as compared with 3H-proline levels measured in Ang II-treated cells infected with mutated shRNAs. B) Expression of AKAP-Lbc and actin in the lysates of infected rat AVFs was assessed by Western blot using specific antibodies, as indicated.

To address this aspect, we determined whether RhoA inhibition or AKAP-Lbc silencing could affect Ang II-induced AVMyoF migration. Myofibroblasts infected with control lentiviruses, or lentiviruses encoding AKAP-Lbc shRNAs or the Flag-tagged RhoAT19N dominant negative mutant were seeded on transwell membranes with 8 μm pores. After a 24 h incubation period with Ang II at 10−7 M, the number of migrated cells on the underside of the membrane was counted. As shown in Fig. 7A and B, stimulation of AVMyoFs infected with control lentiviruses with Ang-II increased their migratory capacity by 4.5 fold compared to untreated cells. The stimulatory effect of Ang II was impaired by 50% to 67% following AKAP-Lbc silencing and by 88% following RhoA inhibition (Fig. 7A and B). This suggests that the AKAP-Lbc–RhoA pathway contributes to the migratory response of myofibroblasts to Ang II. Based on these results, we determined whether AKAP-Lbc silencing could also influence the invasive properties of cardiac myofibroblasts. To address this aspect, we assessed the ability of AVMyoFs to migrate through a Matrigel basement membrane barrier in response to Ang II. Our results indicate that AKAP-Lbc silencing reduces AVMyoF invasion by 55% to 62% compared to cells infected with control lentiviruses or lentiviruses encoding mutated AKAP-Lbc shRNAs (Fig. 7C and D). Collectively, these findings suggest that the AKAP-Lbc–RhoA pathway plays a prominent role in mediating the effects of Ang II on cardiac myofibroblast migration and invasion. 4. Discussion While the implication of Rho signaling in cardiac fibrosis has been suggested by several recent studies [7,9,12,13], the molecular mechanisms that promote Rho activation during this pathological cardiac event are yet to be identified. Our current results demonstrate that the Rho-specific exchange factor AKAP-Lbc is crucially involved in the transduction of pro-fibrotic signals induced by Ang II in cardiac fibroblasts. We initially provided evidence that AKAP-Lbc mediates the effects of Ang II on RhoA

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activation in rat AVFs. Ang II promotes activation of the AKAP-Lbc/ RhoA complex through a signaling pathway that requires the AT1R and the α subunit of the heterotrimeric G protein G12. Importantly, suppression of AKAP-Lbc expression inhibits the ability of Ang II to stimulate multiple pro-fibrotic responses including the expression of the profibrotic factor TGF-β1, expression of α-SMA positive stress fibers (a response that is indicative of the differentiation of quiescent cardiac fibroblast into myofibroblasts), collagen production and myofibroblast migration. Based on these results, we propose a model in which activation of the Gα12–AKAP-Lbc–RhoA pathway downstream of AT1Rs participates in the process of myofibroblast differentiation and favors associated enhancement of collagen synthesis and cell migration (Fig. 8). Collectively, these findings identify AKAP-Lbc as a key regulator of pro-fibrotic responses in adult cardiac fibroblasts. Our previous findings indicate that, in cardiomyocytes, α1-ARs promote cardiomyocyte hypertrophy by stimulating the Rho-GEF activity of AKAP-Lbc through Gα12 [15]. We now show that the Gα12–AKAPLbc–RhoA transduction pathway is also mobilized by Ang II in cardiac fibroblasts to modulate pro-fibrotic responses. This implies that Gα12coupled receptors can activate this conserved AKAP-Lbc-based signaling module to promote pathophysiological responses in both cardiac myocytes and fibroblasts. One intriguing observation is that the contribution of AKAP-Lbc to the activation of RhoA induced by Ang II appears to be influenced by

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Fig. 7. Silencing of AKAP-Lbc expression impairs Ang-II-induced ventricular myofibroblast motility and invasion. A) Adult rat ventricular myofibroblasts infected with control lentiviruses, lentiviruses encoding wild type or mutated AKAP-Lbc shRNAs or with lentiviruses encoding the Flag-RhoA N19 mutant at a MOI of 50, were seeded on transwell membranes with 8 μm pores. After a 24 h incubation period with Ang II at 10−7 M, membranes were fixed in ethanol and cells migrated on the underside of the membrane were visualized using 2% crystal violet. Representative fields for each condition are shown. B) The graph shows the percentage of migrated cells on a total of 10 fields per condition. C) Adult rat ventricular myofibroblasts infected with control lentiviruses or lentiviruses encoding wild type or mutated AKAP-Lbc shRNAs at a MOI of 50, were seeded on transwell membranes with 8 μm pores coated with Matrigel. After an 8 h incubation period with Ang II at 10−7 M, membranes were fixed in ethanol and cells on the underside of the membrane were visualized using 2% crystal violet. Representative fields for each condition are shown. D) The graph shows the percentage of invaded cells on a total of 10 fields per condition. *P b 0.05 as compared with the percentage of migrated (B) or invaded (D) cells infected with lentiviruses encoding for AKAP-Lbc mutated shRNAs and treated with Ang II.

the differentiation state of cardiac fibroblasts (Figs. 1 and S4A). In fact, our results indicate that silencing of AKAP-Lbc expression reduces the ability of Ang II to activate RhoA to a greater extent in cardiac fibroblasts compared to myofibroblasts (100% vs. 51% of inhibition). This raises the possibility that, following differentiation, AKAP-Lbc might regulate RhoA activation in concert with other Rho-GEFs. Possible candidates might include Gα12/13 activated Rho-GEFs such as p115-Rho-GEF,

PDZ-Rho-GEF or LARG, which have been previously shown to mediate various cellular responses to Ang II [33–36]. Our current results provide evidence that AKAP-Lbc controls α-SMA expression and stress fiber formation associated with the process of cardiac myofibroblast differentiation (Figs. 3 and 4). Given the nearly complete inhibition of α-SMA expression observed in AKAP-Lbc silenced fibroblasts, one could suggest that this anchoring protein represents

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that RhoA can regulate collagen I promoter activity through the activation of Smad proteins [41], thus providing a link between RhoA activation and collagen synthesis. Cardiac myofibroblasts display increased migratory and invasive properties, which allow them to reach the sites of injury to promote wound healing and adaptive remodeling [1]. In this respect, previous evidence indicates that RhoA can promote cardiac fibroblast invasion by regulating cell migration as well as the expression and secretion of the matrix metalloproteinase 9 (MMP9) [11], a gelatinase that degrades the basement membrane matrix. Our current results indicate that AKAP-Lbc silencing reduces myofibroblast migration and invasion (Fig. 7) without affecting MMP9 expression (see Fig. S6C, in Supplementary material). These findings are consistent with the idea that inhibition of myofibroblast migration following AKAP-Lbc silencing can negatively impact the ability of myofibroblasts to invade the ECM. In conclusion, based on our current findings highlighting the role of AKAP-Lbc as a key modulator of profibrotic responses in cardiac fibroblasts and on previous evidence demonstrating a role for AKAP-Lbc in cardiomyocyte hypertrophy [15,16,26,42,43] it emerges that targeting the AKAP-Lbc and its ability to bind and activate RhoA could represent a potential strategy for reducing myocardial remodeling in response to cardiac stress. Acknowledgements

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Fig. 8. Model showing the role of AKAP-Lbc in the regulation of pro-fibrotic responses in ventricular fibroblasts. The Rho-GEF activity of AKAP-Lbc is induced in response to Ang II stimulation through a Gα12-mediated signaling pathway. GTP-bound Rho is released from AKAP-Lbc and interacts with downstream effectors to induce α-SMA expression and associated differentiation of quiescent cardiac fibroblasts to myofibroblasts, collagen production, TGF-β1 transcription, and myofibroblast migration.

the main Rho-GEF participating in the process of cardiac fibroblast differentiation induced by Ang II. The fact that expression of the AKAPLbc Y2153F mutant in silenced AVFs is able to rescue the stimulatory effects of Ang II on α-SMA expression (Fig. 3D) strongly suggests that RhoA acts downstream of AKAP-Lbc to promote cardiac myofibroblast differentiation. A RhoA dependent signaling pathway controlling α-SMA gene transcription has been recently identified in cardiac fibroblasts and involves actin polymerization and subsequent nuclear localization of the transcription factors MRTF-A and SRF, which can directly bind the promoter region of the α-SMA gene [8,9]. Another potential alternative effector cascade linking the AKAP-Lbc and RhoA to the transcription of the α-SMA gene is represented by the p38 signaling pathway, which can be selectively activated the AKAP-Lbc complex [25] and which has recently been shown to mediate several profibrotic responses induced by Ang II [37,38]. Interestingly, recent studies indicate that the RhoA specific exchange factor GEFH1/Lfc mediates α-SMA expression in retinal pigment epithelial cells to promote retinal fibrosis [39]. While these findings confirm the notion that RhoA-activating proteins act as key upstream regulators of α-SMA expression, they also suggest that different Rho-GEFs can be mobilized in different cell types to promote α-SMA expression, myofibroblast differentiation and associated fibrosis. Differentiated cardiac myofibroblasts secrete higher amounts of ECM that contribute to cardiac diastolic dysfunction. Previous evidence indicates that inhibition of Rho signaling strongly attenuates interstitial collagen I deposition in the myocardium of mice submitted to pressure overload [40]. We now show, that silencing of the RhoA selective GEF AKAP-Lbc regulates collagen production in adult cardiac fibroblasts (Fig. 5) suggesting a crucial role for AKAP-Lbc–RhoA pathway in the regulation of ECM synthesis. Interestingly, additional evidence suggests

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