A perinuclear calcium compartment regulates cardiac myocyte hypertrophy.

The pleiotropic Ca/calmodulin-dependent phosphatase calcineurin is a key regulator of pathological cardiac myocyte hypertrophy. The selective activation of hypertrophic calcineurin signaling under stress conditions has been attributed to compartmentation of Ca signaling in cardiac myocytes. Here, perinuclear signalosomes organized by the scaffold protein muscle A-Kinase Anchoring Protein β (mAKAPβ/AKAP6β) are shown to orchestrate local Ca transients, inducing calcineurin-dependent NFATc nuclear localization and myocyte hypertrophy in response to β-adrenergic receptor activation.

Subcellular Propagation of Cardiomyocyte β-Adrenergic Activation of Calcium Uptake Involves Internal β-Receptors and AKAP7.

β-adrenergic receptor (β-AR) signaling in cardiac myocytes is central to cardiac function, but spatiotemporal activation within myocytes is unresolved. In rabbit ventricular myocytes, β-AR agonists or high extracellular [Ca] were applied locally at one end, to measure β-AR signal propagation as Ca-transient (CaT) amplitude and sarcoplasmic reticulum (SR) Ca uptake. High local [Ca], increased CaT amplitude under the pipette faster than did ISO, but was also more spatially restricted.

Targeting mAKAPβ expression as a therapeutic approach for ischemic cardiomyopathy.

Ischemic cardiomyopathy is a leading cause of death and an unmet clinical need. Adeno-associated virus (AAV) gene-based therapies hold great promise for treating and preventing heart failure. Previously we showed that muscle A-kinase Anchoring Protein β (mAKAPβ, AKAP6β), a scaffold protein that organizes perinuclear signalosomes in the cardiomyocyte, is a critical regulator of pathological cardiac hypertrophy.

cAMP at Perinuclear mAKAPα Signalosomes Is Regulated by Local Ca Signaling in Primary Hippocampal Neurons.

The second messenger cyclic adenosine monophosphate (cAMP) is important for the regulation of neuronal structure and function, including neurite extension. A perinuclear cAMP compartment organized by the scaffold protein muscle A-kinase anchoring protein α (mAKAPα/AKAP6α) is sufficient and necessary for axon growth by rat hippocampal neurons Here, we report that cAMP at mAKAPα signalosomes is regulated by local Ca signaling that mediates activity-dependent cAMP elevation within that compartment. Simultaneous Forster resonance energy transfer (FRET) imaging using the protein kinase A (PKA) activity reporter AKAR4 and intensiometric imaging using the RCaMP1h fluorescent Ca sensor revealed that membrane depolarization by KCl selectively induced activation of perinuclear PKA activity.

Signalosome-Regulated Serum Response Factor Phosphorylation Determining Myocyte Growth in Width Versus Length as a Therapeutic Target for Heart Failure.

Background: Concentric and eccentric cardiac hypertrophy are associated with pressure and volume overload, respectively, in cardiovascular disease both conferring an increased risk of heart failure. These contrasting forms of hypertrophy are characterized by asymmetrical growth of the cardiac myocyte in mainly width or length, respectively. The molecular mechanisms determining myocyte preferential growth in width versus length remain poorly understood.

mAKAPβ signalosomes - A nodal regulator of gene transcription associated with pathological cardiac remodeling.

Striated myocytes compose about half of the cells of the heart, while contributing the majority of the heart's mass and volume. In response to increased demands for pumping power, including in diseases of pressure and volume overload, the contractile myocytes undergo non-mitotic growth, resulting in increased heart mass, i.e.

Muscle A-kinase-anchoring protein-β-bound calcineurin toggles active and repressive transcriptional complexes of myocyte enhancer factor 2D.

Myocyte enhancer factor 2 (MEF2) transcription factors are key regulators of the development and adult phenotype of diverse tissues, including skeletal and cardiac muscles. Controlled by multiple post-translational modifications, MEF2D is an effector for the Ca/calmodulin-dependent protein phosphatase calcineurin (CaN, PP2B, and PPP3). CaN-catalyzed dephosphorylation promotes the desumoylation and acetylation of MEF2D, increasing its transcriptional activity.

Calcineurin-AKAP interactions: therapeutic targeting of a pleiotropic enzyme with a little help from its friends.

The ubiquitous Ca /calmodulin-dependent phosphatase calcineurin is a key regulator of pathological cardiac hypertrophy whose therapeutic targeting in heart disease has been elusive due to its role in other essential biological processes. Calcineurin is targeted to diverse intracellular compartments by association with scaffold proteins, including by multivalent A-kinase anchoring proteins (AKAPs) that bind protein kinase A and other important signalling enzymes determining cardiac myocyte function and phenotype. Calcineurin anchoring by AKAPs confers specificity to calcineurin function in the cardiac myocyte.

Bidirectional regulation of HDAC5 by mAKAPβ signalosomes in cardiac myocytes.

Class IIa histone deacetylases (HDACs) are transcriptional repressors whose nuclear export in the cardiac myocyte is associated with the induction of pathological gene expression and cardiac remodeling. Class IIa HDACs are regulated by multiple, functionally opposing post-translational modifications, including phosphorylation by protein kinase D (PKD) that promotes nuclear export and phosphorylation by protein kinase A (PKA) that promotes nuclear import. We have previously shown that the scaffold protein muscle A-kinase anchoring protein β (mAKAPβ) orchestrates signaling in the cardiac myocyte required for pathological cardiac remodeling, including serving as a scaffold for both PKD and PKA.

Cushing's syndrome mutant PKA exhibits altered substrate specificity.

The PKA hotspot mutation has been implicated in Cushing's syndrome through hyperactive gain-of-function PKA signaling; however, its influence on substrate specificity has not been investigated. Here, we employ the Proteomic Peptide Library (ProPeL) approach to create high-resolution models for PKA and PKA substrate specificity. We reveal that the L205R mutation reduces canonical hydrophobic preference at the substrate P + 1 position, and increases acidic preference in downstream positions.

Analysis of AKAP7γ Dimerization.

A-kinase anchoring proteins (AKAPs) constitute a family of scaffolding proteins that contribute to spatiotemporal regulation of PKA-mediated phosphorylation events. In particular, AKAP7 is a family of alternatively spliced proteins that participates in cardiac calcium dynamics. Here, we demonstrate via pull-down from transfected cells and by direct protein-protein association that AKAP7γ self-associates.

Phosphorylation state-dependent interaction between AKAP7δ/γ and phospholamban increases phospholamban phosphorylation.

Changes in heart rate and contractility in response to sympathetic stimulation occur via activation of cAMP dependent protein kinase A (PKA), leading to phosphorylation of numerous substrates that alter Ca(2+) cycling. Phosphorylation of these substrates is coordinated by A-kinase anchoring proteins (AKAPs), which recruit PKA to specific substrates [1]. Phosphorylation of the PKA substrate phospholamban (PLB) is a critical determinant of Ca(2+) re-entry into the sarcoplasmic reticulum and is coordinated by AKAP7δ/γ [2,3].

RSK3: A regulator of pathological cardiac remodeling.

The family of p90 ribosomal S6 kinases (RSKs) are pleiotropic effectors for extracellular signal-regulated kinase signaling pathways. Recently, RSK3 was shown to be important for pathological remodeling of the heart. Although cardiac myocyte hypertrophy can be compensatory for increased wall stress, in chronic heart diseases, this nonmitotic cell growth is usually associated with interstitial fibrosis, increased cell death, and decreased cardiac function.

P2X4 receptor-eNOS signaling pathway in cardiac myocytes as a novel protective mechanism in heart failure.

We have demonstrated using immunoprecipitation and immunostaining a novel physical association of the P2X4 receptor (P2X4R), a ligand-gated ion channel, with the cardioprotective, calcium-dependent enzyme endothelial nitric oxide synthase (eNOS). Treatment of murine ventricular myocytes with the P2XR agonist 2-methylthioATP (2-meSATP) to induce a current (mainly Na(+)) increased the formation of nitric oxide (NO), as measured using a fluorescent probe. Possible candidates for downstream effectors mediating eNOS activity include cyclic GMP and PKG or cellular protein nitrosylation.

mAKAP-a master scaffold for cardiac remodeling.

Cardiac remodeling is regulated by an extensive intracellular signal transduction network. Each of the many signaling pathways in this network contributes uniquely to the control of cellular adaptation. In the last few years, it has become apparent that multimolecular signaling complexes or "signalosomes" are important for fidelity in intracellular signaling and for mediating crosstalk between the different signaling pathways.

The scaffold protein muscle A-kinase anchoring protein β orchestrates cardiac myocyte hypertrophic signaling required for the development of heart failure.

Background: Cardiac myocyte hypertrophy is regulated by an extensive intracellular signal transduction network. In vitro evidence suggests that the scaffold protein muscle A-kinase anchoring protein β (mAKAPβ) serves as a nodal organizer of hypertrophic signaling. However, the relevance of mAKAPβ signalosomes to pathological remodeling and heart failure in vivo remains unknown.

Novel protective role of endogenous cardiac myocyte P2X4 receptors in heart failure.

Background: Heart failure (HF), despite continuing progress, remains a leading cause of mortality and morbidity. P2X4 receptors (P2X4R) have emerged as potentially important molecules in regulating cardiac function and as potential targets for HF therapy. Transgenic P2X4R overexpression can protect against HF, but this does not explain the role of native cardiac P2X4R.

Scaffold state switching amplifies, accelerates, and insulates protein kinase C signaling.

Scaffold proteins localize two or more signaling enzymes in close proximity to their downstream effectors. A-kinase-anchoring proteins (AKAPs) are a canonical family of scaffold proteins known to bind protein kinase A (PKA) and other enzymes. Several AKAPs have been shown to accelerate, amplify, and specify signal transduction to dynamically regulate numerous cellular processes.

Regulation of MEF2 transcriptional activity by calcineurin/mAKAP complexes.

The calcium/calmodulin-dependent protein phosphatase calcineurin is required for the induction of transcriptional events that initiate and promote myogenic differentiation. An important effector for calcineurin in striated muscle is the transcription factor myocyte enhancer factor 2 (MEF2). The targeting of the enzyme and substrate to specific intracellular compartments by scaffold proteins often confers specificity in phosphatase activity.

Anchored p90 ribosomal S6 kinase 3 is required for cardiac myocyte hypertrophy.

Rationale: Cardiac myocyte hypertrophy is the main compensatory response to chronic stress on the heart. p90 ribosomal S6 kinase (RSK) family members are effectors for extracellular signal-regulated kinases that induce myocyte growth. Although increased RSK activity has been observed in stressed myocytes, the functions of individual RSK family members have remained poorly defined, despite being potential therapeutic targets for cardiac disease.

Spatiotemporal regulation of PKC via interactions with AKAP7 isoforms.

The regulation of kinases by scaffolding proteins greatly contributes to the fidelity of signal transduction. In the present study, we explored an interaction between the ubiquitous enzyme PKC (protein kinase C) and the scaffolding protein AKAP7 (A-kinase-anchoring protein 7). Using protein biochemistry and surface plasmon resonance approaches, we demonstrate that both AKAP7γ and AKAP7α are capable of high-affinity interactions with multiple isoenzymes of PKC.

Myocyte enhancer factor 2 (MEF2) tethering to muscle selective A-kinase anchoring protein (mAKAP) is necessary for myogenic differentiation.

Differentiation of skeletal myoblast cells to functional myotubes involves highly regulated transcriptional dynamics. The myocyte enhancer factor 2 (MEF2) transcription factors are critical to this process, synergizing with the master regulator MyoD to promote muscle specific gene transcription. MEF2 is extensively regulated by myogenic stimuli, both transcriptionally and post-translationally, but to date there has been little progress in understanding how signals upstream of MEF2 are coordinated to produce a coherent response.

LKB1 destabilizes microtubules in myoblasts and contributes to myoblast differentiation.

Background: Skeletal muscle myoblast differentiation and fusion into multinucleate myotubes is associated with dramatic cytoskeletal changes. We find that microtubules in differentiated myotubes are highly stabilized, but premature microtubule stabilization blocks differentiation. Factors responsible for microtubule destabilization in myoblasts have not been identified.

A-kinase anchoring proteins: scaffolding proteins in the heart.

The pleiotropic cyclic nucleotide cAMP is the primary second messenger responsible for autonomic regulation of cardiac inotropy, chronotropy, and lusitropy. Under conditions of prolonged catecholaminergic stimulation, cAMP also contributes to the induction of both cardiac myocyte hypertrophy and apoptosis. The formation of localized, multiprotein complexes that contain different combinations of cAMP effectors and regulatory enzymes provides the architectural infrastructure for the specialization of the cAMP signaling network.