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ADAP1 limits neonatal cardiomyocyte hypertrophy by reducing integrin cell surface expression.

ABSTRACT: The ArfGAP with dual PH domains 1 (ADAP1) regulates the activation of the hypertrophic mitogen-activated protein kinase ERK1/2 pathway in non-cardiomyocytes. However, its role in cardiomyocytes is unknown. Our aim was to characterize the role of ADAP1 in the hypertrophic process of cardiomyocytes. We assessed the expression of ADAP1 in the hearts of adult and neonatal rats by RT-qPCR and Western blotting and showed that it is preferentially expressed in cardiomyocytes. Adenoviral-mediated ADAP1 overexpression in cultured rat neonatal ventricular cardiomyocytes limited their serum-induced hypertrophic response as measured by immunofluorescence microscopy. Furthermore, ADAP1 overexpression completely blocked phenylephrine- and Mek1 constitutively active (Mek1ca) mutant-induced hypertrophy in these cells. The anti-hypertrophic effect of ADAP1 was not caused by a reduction in protein synthesis, interference with the Erk1/2 pathway, or disruption of the fetal gene program activation, as assessed by nascent protein labeling, Western blotting, and RT-qPCR, respectively. An analysis of cultured cardiomyocytes by confocal microscopy revealed that ADAP1 partially re-organizes ?-actinin into dense puncta, a phenomenon that is synergized by Mek1ca overexpression. Biotin labeling of cell surface proteins from cardiomyocytes overexpressing ADAP1 revealed that it reduces the surface expression of ?1-integrin, an effect that is strongly potentiated by Mek1ca overexpression. Our findings provide insights into the anti-hypertrophic function of ADAP1 in cardiomyocytes.

SUBMITTER: Giguere H

PROVIDER: S-EPMC6134004 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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ADAP1 limits neonatal cardiomyocyte hypertrophy by reducing integrin cell surface expression.

Giguère Hugo H Dumont Audrey-Ann AA Berthiaume Jonathan J Oliveira Vanessa V Laberge Gino G Auger-Messier Mannix M

Scientific reports 20180911 1

The ArfGAP with dual PH domains 1 (ADAP1) regulates the activation of the hypertrophic mitogen-activated protein kinase ERK1/2 pathway in non-cardiomyocytes. However, its role in cardiomyocytes is unknown. Our aim was to characterize the role of ADAP1 in the hypertrophic process of cardiomyocytes. We assessed the expression of ADAP1 in the hearts of adult and neonatal rats by RT-qPCR and Western blotting and showed that it is preferentially expressed in cardiomyocytes. Adenoviral-mediated ADAP1 ...[more]

PMID: 30206251

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Project description:Calreticulin presentation on the cell surface is an important hallmark of immunogenic cell death (ICD), serving as the prophagocytic signal for macrophages. Cell adhesion is a physiologically relevant stimulus previously shown to increase calreticulin interaction with ?-integrins via the juxtamembrane, cytosolic GFFKR motif. This study assessed whether integrin function can regulate surface calreticulin levels in ICD. We generated calreticulin-null T-lymphoblasts and confirmed the loss of surface calreticulin expression on cells treated with doxorubicin, an ICD inducer. Reconstituted expression with full-length calreticulin targeted to the endoplasmic reticulum (ER) successfully rescued doxorubicin-induced surface calreticulin. Reconstitution with a truncation mutant calreticulin targeted to the cytosol led to constitutively high surface calreticulin that was not further elevated by doxorubicin, suggesting calreticulin released from the stressed ER transits the cytosol before its translocation to the cell surface. When stimulated to engage integrin substrates, doxorubicin-treated wild-type T-lymphoblasts exhibited decreased surface calreticulin compared with cells under non-adherent conditions. The inhibitory effect on surface calreticulin was recapitulated for cells in suspension treated with a ?1-integrin-activating antibody, 9EG7. Similarly, cells expressing a truncated ?-integrin cytosolic tail, bearing only the juxtamembrane GFFKR calreticulin-binding motif, exhibited low surface calreticulin with doxorubicin treatment under non-adherent conditions. Using partial permeabilization techniques to distinguish between cytosolic and ER staining, we found that ICD inducers promoted the accumulation of cytosolic calreticulin with negligible change in total calreticulin, suggesting that integrin-mediated inhibition of surface calreticulin was due to reduced cytosolic to surface translocation. T-lymphoblasts co-treated with an ICD inducer and 9EG7 exhibited reduced phagocytosis by macrophages when compared with treatment with only ICD inducer. This study reveals a previously uncharacterized function of integrins as negative regulators of ICD by suppressing presentation of cell surface calreticulin.

Project description:BackgroundRecently shown to regulate cardiac development, the secreted axon guidance molecule SLIT3 maintains its expression in the postnatal heart. Despite its known expression in the cardiovascular system after birth, SLIT3's relevance to cardiovascular function in the postnatal state remains unknown. As such, the objectives of this study were to determine the postnatal myocardial sources of SLIT3 and to evaluate its functional role in regulating the cardiac response to pressure overload stress.MethodsWe performed in vitro studies on cardiomyocytes and myocardial tissue samples from patients and performed in vivo investigation with SLIT3 and ROBO1 (roundabout homolog 1) mutant mice undergoing transverse aortic constriction to establish the role of SLIT3-ROBO1 in adverse cardiac remodeling.ResultsWe first found that SLIT3 transcription was increased in myocardial tissue obtained from patients with congenital heart defects that caused ventricular pressure overload. Immunostaining of hearts from WT (wild-type) and reporter mice revealed that SLIT3 is secreted by cardiac stromal cells, namely fibroblasts and vascular mural cells, within the heart. Conditioned media from cardiac fibroblasts and vascular mural cells both stimulated cardiomyocyte hypertrophy in vitro, an effect that was partially inhibited by an anti-SLIT3 antibody. Also, the N-terminal, but not the C-terminal, fragment of SLIT3 and the forced overexpression of SLIT3 stimulated cardiomyocyte hypertrophy and the transcription of hypertrophy-related genes. We next determined that ROBO1 was the most highly expressed roundabout receptor in cardiomyocytes and that ROBO1 mediated SLIT3's hypertrophic effects in vitro. In vivo, Tcf21+ fibroblast and Tbx18+ vascular mural cell-specific knockout of SLIT3 in mice resulted in decreased left ventricular hypertrophy and cardiac fibrosis after transverse aortic constriction. Furthermore, α-MHC+ cardiomyocyte-specific deletion of ROBO1 also preserved left ventricular function and abrogated hypertrophy, but not fibrosis, after transverse aortic constriction.ConclusionsCollectively, these results indicate a novel role for the SLIT3-ROBO1-signaling axis in regulating postnatal cardiomyocyte hypertrophy induced by pressure overload.

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ADAP1 limits neonatal cardiomyocyte hypertrophy by reducing integrin cell surface expression.

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ADAP1 limits neonatal cardiomyocyte hypertrophy by reducing integrin cell surface expression.

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