Original article
The A-kinase anchor protein AKAP121 is a negative regulator of cardiomyocyte hypertrophy

https://doi.org/10.1016/j.yjmcc.2009.01.018Get rights and content

Abstract

Pathologic cardiac hypertrophy imposes a significant clinical burden on patients, yet the precise intracellular mechanisms responsible for its induction are only partially understood. We examined a potential role for AKAP121 to regulate cardiomyocyte hypertrophy, since recent reports have implicated other AKAPs in this process. We report here that knockdown of AKAP121 expression in isolated neonatal rat cardiomyocytes results in pronounced cellular hypertrophy. Loss of AKAP121 expression is associated with dephosphorylation and nuclear localization of NFATc3, a downstream effector of the hypertrophic phosphatase calcineurin. We also demonstrate that over-expression of AKAP121 in cardiac myocytes reduces basal cell size, and blocks hypertrophy induced by isoproterenol, indicating that AKAP121 negatively regulates the hypertrophic process. Co-immunoprecipitation data indicates that AKAP121 and calcineurin directly interact. Our findings are consistent with a model in which loss of AKAP121 expression leads to the release of an active pool of calcineurin, in turn causing nuclear translocation of NFATc3 and activation of the hypertrophic gene program. These results are the first to identify AKAP121 as a negative regulator of cardiomyocyte hypertrophy, and highlight AKAP121 as a potential target for therapeutic exploitation.

Introduction

Hypertrophy of cardiac myocytes occurs not only during normal development and growth of the fetal and neonatal heart, but also during pathologic remodeling of the adult heart in response to stresses such as hypertension or infarct [1]. Over the past fifteen years, a variety of transcription factors and intracellular signaling pathways have been shown to play key roles in driving cardiac hypertrophic gene expression. These include the calcium-regulated serine/threonine protein phosphatase calcineurin and its downstream effectors, the nuclear factor of activated T cells (NFAT) family of transcription factors which play central roles in pathologic hypertrophy [2], [3]. Other regulators of hypertrophic gene expression and growth include the MEF2 transcription factors and kinases such as ERK5 and PKD1 [4], [5], [6]. Despite the importance of hypertrophic growth in cardiac function and dysfunction, signaling mechanisms regulating this process remain incompletely understood, and it is likely that other important players remain to be identified.

Recently, the A-kinase anchor proteins (AKAPs) have been scrutinized as potential regulators of cardiac hypertrophy. It has been reported that mAKAP/AKAP6 regulates hypertrophy of neonatal rat cardiomyocytes. mAKAP forms a complex with ERK5, which induces a hypertrophic program in response to leukemia inhibitory factor. Knockdown of mAKAP expression inhibited the hypertrophic response, demonstrating the importance of mAKAP in this process [6]. More recently, this same group demonstrated that AKAP-lbc/AKAP13 also regulates cardiomyocyte hypertrophy by forming a complex with PKC and PKD. This complex facilitates PKD activation, leading to downstream MEF2-mediated cardiac hypertrophic gene expression. AKAP-lbc expression is induced in phenylephrine-mediated cardiomyocyte hypertrophy, and knockdown of AKAP-lbc resulted in a decrease in cardiomyocyte size [7]. Conversely, over-expression of AKAP-lbc induced MEF2-mediated reporter gene expression.

Other AKAPs have been reported to be expressed in the heart, including AKAP350/AKAP9, AKAP95/AKAP8 and AKAP121/AKAP1, but whether these AKAPs play a role in regulating cardiac hypertrophy remains unknown [8], [9]. We examined whether AKAP121 can regulate cardiomyocyte hypertrophy via loss-of-function experiments in isolated neonatal rat cardiomyocytes using small hairpin RNA (shRNA) interference. We report here that knockdown of AKAP121 expression results in significant hypertrophy of cardiomyocytes. AKAP121 knockdown also resulted in a net translocation of NFATc3 from the cytosol to the nucleus as demonstrated by immunofluorescence staining. Western blotting revealed that cytosolic levels of phosphorylated NFATc3 decreased, while total nuclear NFATc3 increased, consistent with activation of the calcineurin/NFAT hypertrophic pathway. Using gain-of-function experiments, we further show that over-expression of AKAP121 in cardiomyocytes decreases cell size and renders the cells resistant to isoproterenol-induced hypertrophy. Co-immunoprecipitation data supports the hypothesis that AKAP121 binds to calcineurin, suggesting that knockdown of AKAP121 leads to the release of an active pool of calcineurin. These findings indicate that AKAP121 plays a previously unappreciated regulatory role in the development of cardiac hypertrophy, and thus may be of therapeutic significance.

Section snippets

Cell culture

Transient transfection studies were carried out in COS7 cells, using Lipofectamine 2000 (Invitrogen) for transfection of cells as per manufacturer's directions. COS7 cells were maintained in 10% fetal bovine serum in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 2 mM l-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin.

Cardiomyocytes were isolated from neonatal rat hearts as previously described [10]. This method employs differential plating to minimize contaminating cell

Results

To perform loss-of-function experiments, we identified two potential shRNA sequences targeting rat AKAP121 (NCBI Accession NM_053665; Table 1, Fig. 1), which we designated 71/2 and 73/4, using Invitrogen's BLOCK-iT RNAi Designer software. To test the efficacy of these sequences in knocking down AKAP121 expression, COS-7 cells were transfected with an expression vector encoding a fusion product of green fluorescent protein (GFP) and mouse S-AKAP84, a truncated splice variant of AKAP121 that

Discussion

The development of cardiac hypertrophy has important physiological and pathophysiological consequences for heart function. Physiological hypertrophy underlies the process of fetal cardiac maturation, and results in improved efficiency of the heart in highly exercised individuals such as elite athletes. In contrast, pathological hypertrophy compromises cardiac function, causing increased patient morbidity and mortality. It is therefore of great clinical interest to identify new regulators of

Acknowledgments

The authors gratefully acknowledge Dr. A. Feliciello (University of Naples) for the S-AKAP84–GFP fusion protein and AKAP121 expression vectors. This project was supported by funding from the Canadian Institutes for Health Research (MOP-67012). M.P.C. is a McDonald Scholar of the Heart and Stroke Foundation of Canada.

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