A molecular switch for neuroprotective astrocyte reactivity.

The intrinsic mechanisms that regulate neurotoxic versus neuroprotective astrocyte phenotypes and their effects on central nervous system degeneration and repair remain poorly understood. Here we show that injured white matter astrocytes differentiate into two distinct C3-positive and C3-negative reactive populations, previously simplified as neurotoxic (A1) and neuroprotective (A2)1,2, which can be further subdivided into unique subpopulations defined by proliferation and differential gene expression signatures. We find the balance of neurotoxic versus neuroprotective astrocytes is regulated by discrete pools of compartmented cyclic adenosine monophosphate derived from soluble adenylyl cyclase and show that proliferating neuroprotective astrocytes inhibit microglial activation and downstream neurotoxic astrocyte differentiation to promote retinal ganglion cell survival. Finally, we report a new, therapeutically tractable viral vector to specifically target optic nerve head astrocytes and show that raising nuclear or depleting cytoplasmic cyclic AMP in reactive astrocytes inhibits deleterious microglial or macrophage cell activation and promotes retinal ganglion cell survival after optic nerve injury. Thus, soluble adenylyl cyclase and compartmented, nuclear- and cytoplasmic-localized cyclic adenosine monophosphate in reactive astrocytes act as a molecular switch for neuroprotective astrocyte reactivity that can be targeted to inhibit microglial activation and neurotoxic astrocyte differentiation to therapeutic effect. These data expand on and define new reactive astrocyte subtypes and represent a step towards the development of gliotherapeutics for the treatment of glaucoma and other optic neuropathies.

Targeting mAKAPß 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 ß (mAKAPß, AKAP6ß), a scaffold protein that organizes perinuclear signalosomes in the cardiomyocyte, is a critical regulator of pathological cardiac hypertrophy. Here, we show that inhibition of mAKAPß expression in stressed adult cardiomyocytes in vitro was cardioprotective, while conditional cardiomyocyte-specific mAKAP gene deletion in mice prevented pathological cardiac remodeling due to myocardial infarction. We developed a new self-complementary serotype 9 AAV gene therapy vector expressing a short hairpin RNA for mAKAPß under the control of a cardiomyocyte-specific promoter (AAV9sc.shmAKAP). This vector efficiently downregulated mAKAPß expression in the mouse heart in vivo. Expression of the shRNA also inhibited mAKAPß expression in human induced cardiomyocytes in vitro. Following myocardial infarction, systemic administration of AAV9sc.shmAKAP prevented the development of pathological cardiac remodeling and heart failure, providing long-term restoration of left ventricular ejection fraction. Our findings provide proof-of-concept for mAKAPß as a therapeutic target for ischemic cardiomyopathy and support the development of a translational pipeline for AAV9sc.shmAKAP for the treatment of heart failure.

Differential expression of PIEZO1 and PIEZO2 mechanosensitive channels in ocular tissues implicates diverse functional roles.

PIEZO1 and PIEZO2 are mechanosensitive ion channels that regulate many important physiological processes including vascular blood flow, touch, and proprioception. As the eye is subject to mechanical stress and is highly perfused, these channels may play important roles in ocular function and intraocular pressure regulation. PIEZO channel expression in the eye has not been well defined, in part due to difficulties in validating available antibodies against PIEZO1 and PIEZO2 in ocular tissues. It is also unclear if PIEZO1 and PIEZO2 are differentially expressed. To address these questions, we used single-molecule fluorescence in situ hybridization (smFISH) together with transgenic reporter mice expressing PIEZO fusion proteins under the control of their endogenous promoters to compare the expression and localization of PIEZO1 and PIEZO2 in mouse ocular tissues relevant to glaucoma. We detected both PIEZO1 and PIEZO2 expression in the trabecular meshwork, ciliary body, and in the ganglion cell layer (GCL) of the retina. Piezo1 mRNA was more abundantly expressed than Piezo2 mRNA in these ocular tissues. Piezo1 but not Piezo2 mRNA was detected in the inner nuclear layer and outer nuclear layer of the retina. Our results suggest that PIEZO1 and PIEZO2 are differentially expressed and may have distinct roles as mechanosensors in glaucoma-relevant ocular tissues.

A Novel Recessive Mutation in SPEG Causes Early Onset Dilated Cardiomyopathy.

Dilated cardiomyopathy (DCM) is a common cause of heart failure and sudden cardiac death. It has been estimated that up to half of DCM cases are hereditary. Mutations in more than 50 genes, primarily autosomal dominant, have been reported. Although rare, recessive mutations are thought to contribute considerably to DCM, especially in young children. Here we identified a novel recessive mutation in the striated muscle enriched protein kinase (SPEG, p. E1680K) gene in a family with nonsyndromic, early onset DCM. To ascertain the pathogenicity of this mutation, we generated SPEG E1680K homozygous mutant human induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) using CRISPR/Cas9-mediated genome editing. Functional studies in mutant iPSC-CMs showed aberrant calcium homeostasis, impaired contractility, and sarcomeric disorganization, recapitulating the hallmarks of DCM. By combining genetic analysis with human iPSCs, genome editing, and functional assays, we identified SPEG E1680K as a novel mutation associated with early onset DCM and provide evidence for its pathogenicity in vitro. Our study provides a conceptual paradigm for establishing genotype-phenotype associations in DCM with autosomal recessive inheritance.

A perinuclear calcium compartment regulates cardiac myocyte hypertrophy.

The pleiotropic Ca2+/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 Ca2+ signaling in cardiac myocytes. Here, perinuclear signalosomes organized by the scaffold protein muscle A-Kinase Anchoring Protein ß (mAKAPß/AKAP6ß) are shown to orchestrate local Ca2+ transients, inducing calcineurin-dependent NFATc nuclear localization and myocyte hypertrophy in response to ß-adrenergic receptor activation. Fluorescent biosensors for Ca2+ and calcineurin and protein kinase A (PKA) activity, both diffusely expressed and localized by nesprin-1α to the nuclear envelope, are used to define an autonomous mAKAPß signaling compartment in adult and neonatal rat ventricular myocytes. Notably, ß-adrenergic-stimulated perinuclear Ca2+ and PKA and CaN activity transients depended upon mAKAPß expression, while Ca2+ elevation and PKA and CaN activity in the cytosol were mAKAPß independent. Buffering perinuclear cAMP and Ca2+ prevented calcineurin-dependent NFATc nuclear translocation and myocyte hypertrophy, without affecting cardiac myocyte contractility. Additional findings suggest that the perinuclear Ca2+ transients were mediated by signalosome-associated ryanodine receptors regulated by local PKA phosphorylation. These results demonstrate the existence of a functionally independent Ca2+ signaling compartment in the cardiac myocyte regulating hypertrophy and provide a premise for targeting mAKAPß signalosomes to prevent selectively cardiac hypertrophy in disease.

Distribution of cardiomyocyte-selective adeno-associated virus serotype 9 vectors in swine following intracoronary and intravenous infusion.

Limited reports exist regarding adeno-associated virus (AAV) biodistribution in swine. This study assessed biodistribution following antegrade intracoronary and intravenous delivery of two self-complementary serotype 9 AAV (AAV9sc) biologics designed to target signaling in the cardiomyocyte considered important for the development of heart failure. Under the control of a cardiomyocyte-specific promoter, AAV9sc.shmAKAP and AAV9sc.RBD express a small hairpin RNA for the perinuclear scaffold protein muscle A-kinase anchoring protein ß (mAKAPß) and an anchoring disruptor peptide for p90 ribosomal S6 kinase type 3 (RSK3), respectively. Quantitative PCR was used to assess viral genome (vg) delivery and transcript expression in Ossabaw and Yorkshire swine tissues. Myocardial viral delivery was 2-5 × 105 vg/µg genomic DNA (gDNA) for both infusion techniques at a dose ∼1013 vg/kg body wt, demonstrating delivery of ∼1-3 viral particles per cardiac diploid genome. Myocardial RNA levels for each expressed transgene were generally proportional to dose and genomic delivery, and comparable with levels for moderately expressed endogenous genes. Despite significant AAV9sc delivery to other tissues, including the liver, neither biologic induced toxic effects as assessed using functional, structural, and circulating cardiac and systemic markers. These results indicate successful targeted delivery of cardiomyocyte-selective viral vectors in swine without negative side effects, an important step in establishing efficacy in a preclinical experimental setting.

Soluble α-klotho and heparin modulate the pathologic cardiac actions of fibroblast growth factor 23 in chronic kidney disease.

Fibroblast growth factor (FGF) 23 is a phosphate-regulating hormone that is elevated in patients with chronic kidney disease and associated with cardiovascular mortality. Experimental studies showed that elevated FGF23 levels induce cardiac hypertrophy by targeting cardiac myocytes via FGF receptor isoform 4 (FGFR4). A recent structural analysis revealed that the complex of FGF23 and FGFR1, the physiologic FGF23 receptor in the kidney, includes soluble α-klotho (klotho) and heparin, which both act as co-factors for FGF23/FGFR1 signaling. Here, we investigated whether soluble klotho, a circulating protein with cardio-protective properties, and heparin, a factor that is routinely infused into patients with kidney failure during the hemodialysis procedure, regulate FGF23/FGFR4 signaling and effects in cardiac myocytes. We developed a plate-based binding assay to quantify affinities of specific FGF23/FGFR interactions and found that soluble klotho and heparin mediate FGF23 binding to distinct FGFR isoforms. Heparin specifically mediated FGF23 binding to FGFR4 and increased FGF23 stimulatory effects on hypertrophic growth and contractility in isolated cardiac myocytes. When repetitively injected into two different mouse models with elevated serum FGF23 levels, heparin aggravated cardiac hypertrophy. We also developed a novel procedure for the synthesis and purification of recombinant soluble klotho, which showed anti-hypertrophic effects in FGF23-treated cardiac myocytes. Thus, soluble klotho and heparin act as independent FGF23 co-receptors with opposite effects on the pathologic actions of FGF23, with soluble klotho reducing and heparin increasing FGF23-induced cardiac hypertrophy. Hence, whether heparin injections during hemodialysis in patients with extremely high serum FGF23 levels contribute to their high rates of cardiovascular events and mortality remains to be studied.

Dock10 Regulates Cardiac Function under Neurohormonal Stress.

Dedicator of cytokinesis 10 (Dock10) is a guanine nucleotide exchange factor for Cdc42 and Rac1 that regulates the JNK (c-Jun N-terminal kinase) and p38 MAPK (mitogen-activated protein kinase) signaling cascades. In this study, we characterized the roles of Dock10 in the myocardium. In vitro: we ablated Dock10 in neonatal mouse floxed Dock10 cardiomyocytes (NMCMs) and cardiofibroblasts (NMCFs) by transduction with an adenovirus expressing Cre-recombinase. In vivo, we studied mice in which the Dock10 gene was constitutively and globally deleted (Dock10 KO) and mice with cardiac myocyte-specific Dock10 KO (Dock10 CKO) at baseline and in response to two weeks of Angiotensin II (Ang II) infusion. In vitro, Dock10 ablation differentially inhibited the α-adrenergic stimulation of p38 and JNK in NMCM and NMCF, respectively. In vivo, the stimulation of both signaling pathways was markedly attenuated in the heart. The Dock10 KO mice had normal body weight and cardiac size. However, echocardiography revealed mildly reduced systolic function, and IonOptix recordings demonstrated reduced contractility and elevated diastolic calcium levels in isolated cardiomyocytes. Remarkably, Dock10 KO, but not Dock10 CKO, exaggerated the pathological response to Ang II infusion. These data suggest that Dock10 regulates cardiac stress-related signaling. Although Dock10 can regulate MAPK signaling in both cardiomyocytes and cardiofibroblasts, the inhibition of pathological cardiac remodeling is not apparently due to the Dock10 signaling in the cardiomyocyte.

Calcineurin Aß-Specific Anchoring Confers Isoform-Specific Compartmentation and Function in Pathological Cardiac Myocyte Hypertrophy.

BACKGROUND: The Ca2+/calmodulin-dependent phosphatase calcineurin is a key regulator of cardiac myocyte hypertrophy in disease. An unexplained paradox is how the ß isoform of the calcineurin catalytic A-subunit (CaNAß) is required for induction of pathological myocyte hypertrophy, despite calcineurin Aα expression in the same cells. It is unclear how the pleiotropic second messenger Ca2+ drives excitation-contraction coupling while not stimulating hypertrophy by calcineurin in the normal heart. Elucidation of the mechanisms conferring this selectivity in calcineurin signaling should reveal new strategies for targeting the phosphatase in disease. METHODS: Primary adult rat ventricular myocytes were studied for morphology and intracellular signaling. New Förster resonance energy transfer reporters were used to assay Ca2+ and calcineurin activity in living cells. Conditional gene deletion and adeno-associated virus-mediated gene delivery in the mouse were used to study calcineurin signaling after transverse aortic constriction in vivo. RESULTS: CIP4 (Cdc42-interacting protein 4)/TRIP10 (thyroid hormone receptor interactor 10) was identified as a new polyproline domain-dependent scaffold for CaNAß2 by yeast 2-hybrid screen. Cardiac myocyte-specific CIP4 gene deletion in mice attenuated pressure overload-induced pathological cardiac remodeling and heart failure. Blockade of CaNAß polyproline-dependent anchoring using a competing peptide inhibited concentric hypertrophy in cultured myocytes; disruption of anchoring in vivo using an adeno-associated virus gene therapy vector inhibited cardiac hypertrophy and improved systolic function after pressure overload. Live cell Förster resonance energy transfer biosensor imaging of cultured myocytes revealed that Ca2+ levels and calcineurin activity associated with the CIP4 compartment were increased by neurohormonal stimulation, but minimally by pacing. Conversely, Ca2+ levels and calcineurin activity detected by nonlocalized Förster resonance energy transfer sensors were induced by pacing and minimally by neurohormonal stimulation, providing functional evidence for differential intracellular compartmentation of Ca2+ and calcineurin signal transduction. CONCLUSIONS: These results support a structural model for Ca2+ and CaNAß compartmentation in cells based on an isoform-specific mechanism for calcineurin protein-protein interaction and localization. This mechanism provides an explanation for the specific role of CaNAß in hypertrophy and its selective activation under conditions of pathologic stress. Disruption of CaNAß polyproline-dependent anchoring constitutes a rational strategy for therapeutic targeting of CaNAß-specific signaling responsible for pathological cardiac remodeling in cardiovascular disease deserving of further preclinical investigation.

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. Identification of the mechanisms governing asymmetrical myocyte growth could provide new therapeutic targets for the prevention or treatment of heart failure. METHODS: Primary adult rat ventricular myocytes, adeno-associated virus (AAV)-mediated gene delivery in mice, and human tissue samples were used to define a regulatory pathway controlling pathological myocyte hypertrophy. Chromatin immunoprecipitation assays with sequencing and precision nuclear run-on sequencing were used to define a transcriptional mechanism. RESULTS: We report that asymmetrical cardiac myocyte hypertrophy is modulated by SRF (serum response factor) phosphorylation, constituting an epigenomic switch balancing the growth in width versus length of adult ventricular myocytes in vitro and in vivo. SRF Ser103 phosphorylation is bidirectionally regulated by RSK3 (p90 ribosomal S6 kinase type 3) and PP2A (protein phosphatase 2A) at signalosomes organized by the scaffold protein mAKAPß (muscle A-kinase anchoring protein ß), such that increased SRF phosphorylation activates AP-1 (activator protein-1)-dependent enhancers that direct myocyte growth in width. AAV are used to express in vivo mAKAPß-derived RSK3 and PP2A anchoring disruptor peptides that block the association of the enzymes with the mAKAPß scaffold. Inhibition of RSK3 signaling prevents concentric cardiac remodeling induced by pressure overload, while inhibition of PP2A signaling prevents eccentric cardiac remodeling induced by myocardial infarction, in each case improving cardiac function. SRF Ser103 phosphorylation is significantly decreased in dilated human hearts, supporting the notion that modulation of the mAKAPß-SRF signalosome could be a new therapeutic approach for human heart failure. CONCLUSIONS: We have identified a new molecular switch, namely mAKAPß signalosome-regulated SRF phosphorylation, that controls a transcriptional program responsible for modulating changes in cardiac myocyte morphology that occur secondary to pathological stressors. Complementary AAV-based gene therapies constitute rationally-designed strategies for a new translational modality for heart failure.

Regulation of Neuronal Survival and Axon Growth by a Perinuclear cAMP Compartment.

cAMP signaling is known to be critical in neuronal survival and axon growth. Increasingly the subcellular compartmentation of cAMP signaling has been appreciated, but outside of dendritic synaptic regulation, few cAMP compartments have been defined in terms of molecular composition or function in neurons. Specificity in cAMP signaling is conferred in large part by A-kinase anchoring proteins (AKAPs) that localize protein kinase A and other signaling enzymes to discrete intracellular compartments. We now reveal that cAMP signaling within a perinuclear neuronal compartment organized by the large multivalent scaffold protein mAKAPα promotes neuronal survival and axon growth. mAKAPα signalosome function is explored using new molecular tools designed to specifically alter local cAMP levels as studied by live-cell FRET imaging. In addition, enhancement of mAKAPα-associated cAMP signaling by isoform-specific displacement of bound phosphodiesterase is demonstrated to increase retinal ganglion cell survival in vivo in mice of both sexes following optic nerve crush injury. These findings define a novel neuronal compartment that confers cAMP regulation of neuroprotection and axon growth and that may be therapeutically targeted in disease.SIGNIFICANCE STATEMENT cAMP is a second messenger responsible for the regulation of diverse cellular processes including neuronal neurite extension and survival following injury. Signal transduction by cAMP is highly compartmentalized in large part because of the formation of discrete, localized multimolecular signaling complexes by A-kinase anchoring proteins. Although the concept of cAMP compartmentation is well established, the function and identity of these compartments remain poorly understood in neurons. In this study, we provide evidence for a neuronal perinuclear cAMP compartment organized by the scaffold protein mAKAPα that is necessary and sufficient for the induction of neurite outgrowth in vitro and for the survival of retinal ganglion cells in vivo following optic nerve injury.

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

The ubiquitous Ca2+ /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. Targeting of calcineurin 'signalosomes' may provide a rationale for inhibiting the phosphatase in disease.

Muscle A-kinase-anchoring protein-ß-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 Ca2+/calmodulin-dependent protein phosphatase calcineurin (CaN, PP2B, and PPP3). CaN-catalyzed dephosphorylation promotes the desumoylation and acetylation of MEF2D, increasing its transcriptional activity. Both MEF2D and CaN bind the scaffold protein muscle A-kinase-anchoring protein ß (mAKAPß), which is localized to the nuclear envelope, such that C2C12 skeletal myoblast differentiation and neonatal rat ventricular myocyte hypertrophy are inhibited by mAKAPß signalosome targeting. Using immunoprecipitation and DNA-binding assays, we now show that the formation of mAKAPß signalosomes is required for MEF2D dephosphorylation, desumoylation, and acetylation in C2C12 cells. Reduced MEF2D phosphorylation was coupled to a switch from type IIa histone deacetylase to p300 histone acetylase binding that correlated with increased MEF2D-dependent gene expression and ventricular myocyte hypertrophy. Together, these results highlight the importance of mAKAPß signalosomes for regulating MEF2D activity in striated muscle, affirming mAKAPß as a nodal regulator in the myocyte intracellular signaling network.

Bidirectional regulation of HDAC5 by mAKAPß 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 ß (mAKAPß) orchestrates signaling in the cardiac myocyte required for pathological cardiac remodeling, including serving as a scaffold for both PKD and PKA. We now show that mAKAPß is a scaffold for HDAC5 in cardiac myocytes, forming signalosomes containing HDAC5, PKD, and PKA. Inhibition of mAKAPß expression attenuated the phosphorylation of HDAC5 by PKD and PKA in response to α- and ß-adrenergic receptor stimulation, respectively. Importantly, disruption of mAKAPß-HDAC5 anchoring prevented the induction of HDAC5 nuclear export by α-adrenergic receptor signaling and PKD phosphorylation. In addition, disruption of mAKAPß-PKA anchoring prevented the inhibition by ß-adrenergic receptor stimulation of α-adrenergic-induced HDAC5 nuclear export. Together, these data establish that mAKAPß signalosomes serve to bidirectionally regulate the nuclear-cytoplasmic localization of class IIa HDACs. Thus, the mAKAPß scaffold serves as a node in the myocyte regulatory network controlling both the repression and activation of pathological gene expression in health and disease, respectively.

RSK3 is required for concentric myocyte hypertrophy in an activated Raf1 model for Noonan syndrome.

Noonan syndrome (NS) is a congenital disorder resulting from mutations of the Ras-Raf signaling pathway. Hypertrophic cardiomyopathy associated with RAF1 "RASopathy" mutations is a major risk factor for heart failure and death in NS and has been attributed to activation of MEK1/2-ERK1/2 mitogen-activated protein kinases. We recently discovered that type 3 p90 ribosomal S6 kinase (RSK3) is an ERK effector that is required, like ERK1/2, for concentric myocyte hypertrophy in response to pathological stress such as pressure overload. In order to test whether RSK3 also contributes to NS-associated hypertrophic cardiomyopathy, RSK3 knock-out mice were crossed with mice bearing the Raf1(L613V) human NS mutation. We confirmed that Raf1(L613V) knock-in confers a NS-like phenotype, including cardiac hypertrophy. Active RSK3 was increased in Raf1(L613V) mice. Constitutive RSK3 gene deletion prevented the Raf1(L613V)-dependent concentric growth in width of the cardiac myocyte and attenuated cardiac hypertrophy in female mice. These results are consistent with RSK3 being an important mediator of ERK1/2-dependent growth in RASopathy. In conjunction with previously published data showing that RSK3 is important for pathological remodeling of the heart, these data suggest that targeting of this downstream MAP-kinase pathway effector should be considered in the treatment of RASopathy-associated hypertrophic cardiomyopathy.

The role of soluble adenylyl cyclase in neurite outgrowth.

Axon regeneration in the mature central nervous system is limited by extrinsic inhibitory signals and a postnatal decline in neurons' intrinsic growth capacity. Neuronal levels of the second messenger cAMP are important in regulating both intrinsic growth capacity and neurons' responses to extrinsic factors. Approaches which increase intracellular cAMP in neurons enhance neurite outgrowth and facilitate regeneration after injury. Thus, understanding the factors which affect cAMP in neurons is of potential therapeutic importance. Recently, soluble adenylyl cyclase (sAC, ADCY10), the ubiquitous, non-transmembrane adenylyl cyclase, was found to play a key role in neuronal survival and axon growth. sAC is activated by bicarbonate and cations and may translate physiologic signals from metabolism and electrical activity into a neuron's decision to survive or regenerate. Here we critically review the literature surrounding sAC and cAMP signaling in neurons to further elucidate the potential role of sAC signaling in neurite outgrowth and regeneration. This article is part of a Special Issue entitled: The role of soluble adenylyl cyclase in health and disease.

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. Although RSK3 is less abundant in the cardiac myocyte than other RSK family members, RSK3 appears to serve a unique role in cardiac myocyte stress responses. A potential mechanism conferring the unique function of RSK3 in the heart is anchoring by the scaffold protein muscle A-kinase anchoring protein ß (mAKAPß). Recent findings suggest that RSK3 should be considered as a therapeutic target for the prevention of heart failure, a clinical syndrome of major public health significance.

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. OBJECTIVE: To demonstrate that type 3 RSK (RSK3) is required for cardiac myocyte hypertrophy. METHODS AND RESULTS: RSK3 contains a unique N-terminal domain that is not conserved in other RSK family members. We show that this domain mediates the regulated binding of RSK3 to the muscle A-kinase anchoring protein scaffold, defining a novel kinase anchoring event. Disruption of both RSK3 expression using RNA interference and RSK3 anchoring using a competing muscle A-kinase anchoring protein peptide inhibited the hypertrophy of cultured myocytes. In vivo, RSK3 gene deletion in the mouse attenuated the concentric myocyte hypertrophy induced by pressure overload and catecholamine infusion. CONCLUSIONS: Taken together, these data demonstrate that anchored RSK3 transduces signals that modulate pathologic myocyte growth. Targeting of signaling complexes that contain select kinase isoforms should provide an approach for the specific inhibition of cardiac myocyte hypertrophy and for the development of novel strategies for the prevention and treatment of heart failure.

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. These complexes integrate upstream signals and control downstream effectors. In the cardiac myocyte, the protein mAKAPß serves as a scaffold for a large signalosome that is responsive to cAMP, calcium, hypoxia, and mitogen-activated protein kinase signaling. The main function of mAKAPß signalosomes is to modulate stress-related gene expression regulated by the transcription factors NFATc, MEF2, and HIF-1α and type II histone deacetylases that control pathological cardiac hypertrophy.

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. We now show that the scaffolding protein mAKAP organizes a calcineurin/MEF2 signaling complex in myocytes, regulating gene transcription. A calcineurin/mAKAP/MEF2 complex can be isolated from C2C12 cells and cardiac myocytes, and the calcineurin/MEF2 association is dependent on mAKAP expression. We have identified a peptide comprising the calcineurin binding domain in mAKAP that can disrupt the binding of the phosphatase to the scaffold in vivo. Dominant interference of calcineurin/mAKAP binding blunts the increase in MEF2 transcriptional activity seen during myoblast differentiation, as well as the expression of endogenous MEF2-target genes. Furthermore, disruption of calcineurin binding to mAKAP in cardiac myocytes inhibits adrenergic-induced cellular hypertrophy. Together these data illustrate the importance of calcineurin anchoring by the mAKAP scaffold for MEF2 regulation.