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. 2011 Aug 1;91(3):456-64.
doi: 10.1093/cvr/cvr103. Epub 2011 Apr 14.

IQGAP1 regulates ERK1/2 and AKT signalling in the heart and sustains functional remodelling upon pressure overload

Mauro Sbroggiò  1 , Daniela Carnevale, Alessandro Bertero, Giuseppe Cifelli, Emanuele De Blasio, Giada Mascio, Emilio Hirsch, Wadie F Bahou, Emilia Turco, Lorenzo Silengo, Mara Brancaccio, Giuseppe Lembo, Guido Tarone
Affiliations

IQGAP1 regulates ERK1/2 and AKT signalling in the heart and sustains functional remodelling upon pressure overload

Mauro Sbroggiò et al. Cardiovasc Res. .

Abstract

Aims: The Raf-MEK1/2-ERK1/2 (ERK1/2-extracellular signal-regulated kinases 1/2) signalling cascade is crucial in triggering cardiac responses to different stress stimuli. Scaffold proteins are key elements in coordinating signalling molecules for their appropriate spatiotemporal activation. Here, we investigated the role of IQ motif-containing GTPase-activating protein 1 (IQGAP1), a scaffold for the ERK1/2 cascade, in heart function and remodelling in response to pressure overload.

Methods and results: IQGAP1-null mice have unaltered basal heart function. When subjected to pressure overload, IQGAP1-null mice initially develop a compensatory hypertrophy indistinguishable from that of wild-type (WT) mice. However, upon a prolonged stimulus, the hypertrophic response develops towards a thinning of left ventricular walls, chamber dilation, and a decrease in contractility, in an accelerated fashion compared with WT mice. This unfavourable cardiac remodelling is characterized by blunted reactivation of the foetal gene programme, impaired cardiomyocyte hypertrophy, and increased cardiomyocyte apoptosis. Analysis of signalling pathways revealed two temporally distinct waves of both ERK1/2 and AKT phosphorylation peaking, respectively, at 10 min and 4 days after aortic banding in WT hearts. IQGAP1-null mice show strongly impaired phosphorylation of MEK1/2-ERK1/2 and AKT following 4 days of pressure overload, but normal activation of these kinases after 10 min. Pull-down experiments indicated that IQGAP1 is able to bind the three components of the ERK cascade, namely c-Raf, MEK1/2, and ERK1/2, as well as AKT in the heart.

Conclusion: These data demonstrate, for the first time, a key role for the scaffold protein IQGAP1 in integrating hypertrophy and survival signals in the heart and regulating long-term left ventricle remodelling upon pressure overload.

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Figures

Figure 1
Figure 1
IQGAP1-null mice develop a cardiac hypertrophy similar to WT mice after 1-week AB. (A) Western blot analysis on protein extracts from cardiac fibroblasts (CF) and cardiomyocytes (CM) from newborn WT mice. β1D integrin and vimentin were used as markers for cardiomyocytes and fibroblasts, respectively. Vinculin was used as a loading control. (B–F) Echocardiographic analysis of WT and IQGAP1-null mice (IQGAP1–/–) in basal conditions and after 1 week of AB. IVSd, diastolic intraventricular septum thickness; LVPWd, diastolic left ventricle posterior wall thickness; LVIDd, diastolic left ventricle inner diameter; RWT, relative wall thickness in diastole; %FS, fractional shortening. *P < 0.05 vs. basal (n= 10 hearts/group).
Figure 2
Figure 2
IQGAP1-null mice showed impaired heart function after 12 weeks of AB. (A–F) Echocardiographic parameters acquired at the indicated time points (weeks of AB) from wild type (WT) and IQGAP1-null mice (IQGAP1–/–) subjected to pressure overload. IVSd, diastolic intraventricular septum thickness; LVPWd, diastolic left ventricle posterior wall thickness; LVIDd, diastolic left ventricle inner diameter; RWT, relative wall thickness in diastole; %FS, fractional shortening. *P < 0.05 between groups (n= 10 hearts/group).
Figure 3
Figure 3
IQGAP1 is required for cardiomyocyte hypertrophy and survival in response to long-standing pressure overload. Hearts from WT and IQGAP1-null mice (IQGAP1−/−), sham-operated or subjected to aortic banding (AB) for 1 or 12 weeks, were analysed. (A) Relative fibrosis measured by picrosirius red staining of histological sections (n= 4 hearts/group). (B) Density of FITC-isolectin-stained capillaries. (C) Leucocyte infiltration measured as number of CD18+ cells (n= 4 hearts/group). (D) Cardiomyocyte cross-sectional area (CSA) (n = 4 hearts/group). (E) Apoptotic TUNEL-positive cardiomyocytes (n = 6 hearts/group for sham and 1-week AB, and n = 4 hearts/group at 12-week AB). (F) Western blot analysis of Bcl-XL, Bad, and p53 from WT and IQGAP1-null hearts after 1 week of AB; GAPDH was used as a loading control. (G) Densitometric quantification of the western blot bands (n = 4 hearts/group). (H) Western blot analysis of Bcl-XL, Bad, and p53 from sham WT and IQGAP1-null hearts; GAPDH was used as a loading control. (I) Densitometric quantification of the western blot bands (n = 6 hearts/group). (J–M) Real-time PCR of ANP, BNP, β-MHC, and α-MHC mRNAs from sham or 1 and 12 weeks of AB (n = 6 hearts/group). (N) Western blot analysis of Bcl-XL, Bad, and p53 from WT and IQGAP1-null hearts after 12 weeks of AB; GAPDH was used as a loading control. (O) Densitometric quantification of the western blot bands (n = 6 hearts/group). °P < 0.05 vs. sham; *P < 0.05; **P < 0.01; ***P < 0.001 #P < 0.05 vs. 1-week AB.
Figure 4
Figure 4
IQGAP1 mediates the second phase of ERK1/2 and AKT phosphorylation in response to pressure overload. (A) Western blot analysis of phosphorylated and total MEK1/2, ERK1/2, and AKT from WT mice subjected to AB for the indicated time. (B) Western blot analysis of phosphorylated and total MEK1/2, ERK1/2, and AKT from WT and IQGAP1-null mice subjected to 10 min of AB. (C) Quantification of western blot bands, expressed as phospho/total protein ratio (n = 6 hearts/group). (D) Western blot analysis of phosphorylated and total MEK1/2, ERK1/2, and AKT in WT and IQGAP1−/− hearts subjected to AB for the indicated time. Vinculin was used as a loading control. (E) Quantification of western blot bands expressed as phospho/total protein ratio (n = 4 hearts/group). *P < 0.05 vs. sham; °P < 0.05 vs. WT.
Figure 5
Figure 5
IQGAP1 interacts with c-Raf, MEK1/2, ERK1/2, and AKT in the heart. (A) MBP-fused recombinant IQGAP1 fragments (N, N-terminal fragment; M, middle fragment; C, C-terminal fragment; IQ, IQ domain-containing fragment; WW, WW domain-containing fragment; RGCT, RGCT domain-containing fragment; RGCT-Δ1, RGCT domain-containing fragment deletion mutant 1; RGCT-Δ2, RGCT domain-containing fragment deletion mutant 2). Columns on the right indicate the capability of each fragment to interact (+) or not (−) with the indicated proteins as evaluated by pull-down assays (nt, not tested). (B and C) Pull-down experiments with MBP-fused IQGAP1 fragments were used to map the c-Raf (B) and AKT (C) binding region on IQGAP1. This interaction was revealed by western blotting (upper panels). Recombinant proteins used in the pull-down assay were visualized by Coomassie staining (lower panel). (D) AKT and IQGAP1co-immunoprecipitation was detected by western blotting. Input: heart total protein extract. Input was analysed in the same blot; however, a lower exposure is shown to allow a precise detection of the reference band. Figures showed representative pull-down and immunoprecipitation analyses that have been performed at least three times with comparable results.
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