Original articleThe C-terminus of the long AKAP13 isoform (AKAP-Lbc) is critical for development of compensatory cardiac hypertrophy
Introduction
Localized regulation and integration of intracellular signal transduction is important for cardiac function. Disruption of appropriate signaling results in the development of heart failure [1], [2]. A-Kinase Anchoring Proteins (AKAPs) are fundamental regulatory molecules involved in signal transduction, functioning to target unique signaling complexes to distinct subcellular locations, thereby coordinating signaling and generating substrate specificity [3]. In the heart, AKAPs play a crucial role by integrating cAMP-dependent protein kinase (protein kinase A; PKA) signaling with additional enzymes to modulate physiological functions, including Ca2 +-cycling and cardiac contractility [4], as well as pathological processes involved in cardiac remodeling and the development of heart failure [5]. Subcellular localization of signaling components by AKAPs is important for cardiac function. Multiple AKAPs have been identified in cardiac myocytes, targeting signaling complexes to distinct subcellular regions including the sarcolemma [6], sarcoplasic reticulum [7], nuclear envelope [8], and sarcomere [9], [10], [11].
A recent proteomic study suggests that differential expression of AKAPs coupled with alterations in the AKAP “interactome” may be critical factors in heart failure [12], however currently, few AKAP knockout or transgenic mouse models have been studied to specifically determine in vivo (patho)physiological roles in healthy and diseased heart. Here, we focus on the in vivo role of the AKAP13 gene long transcript, called AKAP-Lbc; due to an N-terminal A-Kinase Anchoring domain [13] and a C-terminal region originally identified in a screen for transforming genes from human myeloid leukemia patients in Lymphoid Blast Crisis [14]. AKAP-Lbc serves as a scaffold for multiple protein kinases, including PKA, protein kinase C (PKCα and PKCη isoforms) and protein kinase D (PKD1) [15]. AKAP-Lbc also acts as a guanine exchange factor (GEF) for Rho [13] and mediates activation of p38α MAPK [16], ERK1/2 [17] and IκB kinase β (IKKβ) [18]. Additionally, we have recently demonstrated that AKAP-Lbc tethers the tyrosine phosphatase Shp2; which is inhibited by PKA phosphorylation in the AKAP-Lbc complex under hypertrophic conditions in the heart [19]. AKAP-Lbc is predominantly expressed in the heart [13] and is essential for cardiac function. Knockout of AKAP-Lbc in mice leads to embryonic lethality due to decreased expression of cardiac developmental genes and deficient sarcomere formation in developing myocytes, resulting in a thin myocardium in the developing heart [20]. Previously, we and others have demonstrated a role for AKAP-Lbc in the induction of cardiac hypertrophy in vitro [21], [22]. Cardiac myocytes primarily respond to increased workload by an increase in size (hypertrophy). Initially cardiac hypertrophy is a beneficial, compensatory process, decreasing wall stress and increasing cardiac output and stroke volume. However, prolonged hypertrophy is maladaptive, transitioning to decompensation and cardiac failure [23], [24]. Understanding how molecular events are orchestrated by AKAP-Lbc may lead to the identification of new pharmacological approaches for the treatment of heart failure.
AKAP-Lbc expression is upregulated in hypertrophic neonatal rat ventricular myocytes (NRVM), whereas siRNA-silencing of AKAP-Lbc expression reduces phenylephrine (PE)-stimulated expression of hypertrophic markers and hypertrophy [21], [22]. A similar increase in AKAP-Lbc expression was also observed in human heart specimens obtained from patients with hypertrophic cardiomyopathy where AKAP-Lbc mRNA content was increased, compared to control age-matched healthy human heart samples [21].
In knockdown/rescue experiments using NRVM, to dissect signaling through AKAP-Lbc, our results show that AKAP-Lbc scaffolding of PKD1 is the predominant mechanism of AKAP-Lbc-mediated hypertrophy [21]. Mechanistically, AKAP-Lbc facilitates activation of PKD1 (the predominant protein kinase D cardiac isoform [25], [26], [27]) in response to hypertrophic stimuli including PE and endothelin-1 (ET-1). AKAP-Lbc contributes to PKD1 activation in two ways: first, by bringing PKC and PKD1 into close proximity, thereby facilitating phosphorylation and activation of PKD1 by PKC. Second, PKA phosphorylation of AKAP-Lbc, in the PKD1 binding region of AKAP-Lbc (at S2737) releases newly activated PKD1 from the AKAP-Lbc complex. Thus, AKAP-Lbc-anchored PKC and PKA synergistically activate PKD1 by promoting activation and passage of multiple PKD1 molecules through AKAP-Lbc [14].
Activation of PKD1 through AKAP-Lbc facilitates phosphorylation and subsequent nuclear export of histone deacetylase 5 (HDAC5) [21], leading to de-repression of the transcription factor MEF2, resulting in cardiac myocyte hypertrophy through MEF2-mediated transcription of muscle-specific genes and re-expression of developmental genes [28], [29]. Currently, the in vivo role of this signaling pathway is unknown. Therefore, we set out to determine the role of AKAP-Lbc-PKD1 in the context of pathological hypertrophy and the development of heart failure. In this report, we utilize a gene-trap mouse expressing a form of AKAP-Lbc that is truncated at the C-terminus and unable to bind PKD1. AKAP-Lbc-ΔPKD mice are viable, displaying normal cardiac structure and electrocardiograms. AKAP-Lbc-PKD1 signaling does not appear to be critical for development, but may play a minor role under conditions of β-adrenergic (predominantly Gs-Protein-Coupled Receptor)-induced cardiac hypertrophic remodeling. In response to isoproterenol treatment, mice lacking both the GEF and PKD-binding domains of AKAP-Lbc display abnormal cardiac contractility despite a similar increase in heart size, compared to control wild-type (WT) mice [30].
Here, we demonstrate an in vivo role for AKAP-Lbc in the induction of compensatory myocardial hypertrophy in response to pressure overload and angiotensin-II/phenylephrine (AT-II/PE) treatment, both known to activate PKD1, predominantly via Gq-PCR mediated pathways.
Section snippets
Generation of the AKAP-Lbc-ΔPKD mouse
The AKAP-Lbc-ΔPKD mouse was generated from MMRRC gene-trap ES cell line CSJ288 (strain genetic background: B6NCr.129P2) on a C57Bl/6 background and is fully described in [30]. The gene-trap construct uses a strong splice acceptor to create a fused mRNA of upstream AKAP-Lbc exons with the trapping cassette [31]. The AKAP-Lbc-ΔPKD truncation mutant results from specific integration of a β-Geo cassette (β-Galactosidase/neomycin resistance gene) within the endogenous AKAP-Lbc genomic locus,
Characterization of AKAP-Lbc-ΔPKD
Fig. 1A shows a diagram of AKAP-Lbc-ΔPKD, which is expressed in the gene-trap mouse, developed from Mutant Mouse Regional Resource Ceter (MMRRC) gene-trap embryonic stem cell line CSJ288. AKAP-Lbc-ΔPKD refers to a truncated form of AKAP-Lbc that cannot bind PKD1 (as demonstrated by the Western blot in Fig. 1D showing co-immunoprecipitation of endogenous PKD1 with AKAP-Lbc-WT, but not AKAP-Lbc-ΔPKD. Generation and initial characterization of AKAP-Lbc-ΔPKD mice, as well as extensive
Discussion
This study defines an in vivo role for AKAP-Lbc in the regulation of pathological cardiac hypertrophy and remodeling under certain conditions of cardiac stress. Unlike the hypertrophic response induced by isoproterenol, via β-adrenergic signaling [30], AKAP-Lbc-ΔPKD mice fail to develop compensatory cardiac hypertrophy in response to chronic AT-II/PE treatment or TAC-induced pressure overload. Collectively, these results indicate that the C-terminus of AKAP-Lbc, which binds PKD1 and mediates
Conclusion
In summary, we have demonstrated a critical in vivo role for AKAP-Lbc in the development of compensatory hypertrophy, in part, by mediating PKD1 phosphorylation of HDAC5, and promoting hypertrophic gene transcription to enhance cardiac performance in response to TAC-induced pressure overload and AT-II/PE treatment. Functionally, AKAP-Lbc-PKD1 signaling acts to reduce the development of cardiac fibrosis, apoptosis and the development of heart failure.
Sources of funding
This study was supported by the American Heart Association Grant 11SDG5230003 to GKC and the National Center for Advancing Translational Science — UIC Center for Clinical and Translational Sciences Grant UL1TR000050. BTB was supported by the National Institutes of Health (NIH) T32 Training Grant 5T32HL072742-09 through the University of Illinois at Chicago Department of Cardiology. MMM was supported by NIH grants T32 HL07692-16-20 and F32 HL116094. KB was supported by NIH grants HL089617 and
Disclosures
None.
Acknowledgments
We thank Dr. Robert Gaffin (the UIC CCVR Physiology Core) for help with TAC and blood pressure measurements, and Dr. Matthew Curtis (UIC RRC Imaging Core) for help with microscopy.
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