Cardiac Hypertrophy Is Inhibited by a Local Pool of cAMP Regulated by Phosphodiesterase 2.

RATIONALE: Chronic elevation of 3'-5'-cyclic adenosine monophosphate (cAMP) levels has been associated with cardiac remodeling and cardiac hypertrophy. However, enhancement of particular aspects of cAMP/protein kinase A signaling seems to be beneficial for the failing heart. cAMP is a pleiotropic second messenger with the ability to generate multiple functional outcomes in response to different extracellular stimuli with strict fidelity, a feature that relies on the spatial segregation of the cAMP pathway components in signaling microdomains. OBJECTIVE: How individual cAMP microdomains affect cardiac pathophysiology remains largely to be established. The cAMP-degrading enzymes phosphodiesterases (PDEs) play a key role in shaping local changes in cAMP. Here we investigated the effect of specific inhibition of selected PDEs on cardiac myocyte hypertrophic growth. METHODS AND RESULTS: Using pharmacological and genetic manipulation of PDE activity, we found that the rise in cAMP resulting from inhibition of PDE3 and PDE4 induces hypertrophy, whereas increasing cAMP levels via PDE2 inhibition is antihypertrophic. By real-time imaging of cAMP levels in intact myocytes and selective displacement of protein kinase A isoforms, we demonstrate that the antihypertrophic effect of PDE2 inhibition involves the generation of a local pool of cAMP and activation of a protein kinase A type II subset, leading to phosphorylation of the nuclear factor of activated T cells. CONCLUSIONS: Different cAMP pools have opposing effects on cardiac myocyte cell size. PDE2 emerges as a novel key regulator of cardiac hypertrophy in vitro and in vivo, and its inhibition may have therapeutic applications.

Small heat shock protein 20 (Hsp20) facilitates nuclear import of protein kinase D 1 (PKD1) during cardiac hypertrophy.

BACKGROUND: Nuclear import of protein kinase D1 (PKD1) is an important event in the transcriptional regulation of cardiac gene reprogramming leading to the hypertrophic growth response, however, little is known about the molecular events that govern this event. We have identified a novel complex between PKD1 and a heat shock protein (Hsp), Hsp20, which has been implicated as cardioprotective. This study aims to characterize the role of the complex in PKD1-mediated myocardial regulatory mechanisms that depend on PKD1 nuclear translocation. RESULTS: In mapping the Hsp20 binding sites on PKD1 within its catalytic unit using peptide array analysis, we were able to develop a cell-permeable peptide that disrupts the Hsp20-PKD1 complex. We use this peptide to show that formation of the Hsp20-PKD1 complex is essential for PKD1 nuclear translocation, signaling mechanisms leading to hypertrophy, activation of the fetal gene programme and pathological cardiac remodeling leading to cardiac fibrosis. CONCLUSIONS: These results identify a new signaling complex that is pivotal to pathological remodelling of the heart that could be targeted therapeutically.

Heat shock protein 20 (HSP20) is a novel substrate for protein kinase D1 (PKD1).

Heat shock protein 20 (HSP20) has cardioprotective qualities, which are triggered by PKA phosphorylation. PKD1 is also a binding partner for HSP20, and this prompted us to investigate whether the chaperone was a substrate for PKD1. We delineate the PKD1 binding sites on HSP20 and show for the first time HSP20 is a substrate for PKD1. Phosphorylation of HSP20 by PKD1 is diminished by pharmacological or siRNA reduction of PKD1 activity and is enhanced following PKD1 activation. Our results suggest that both PKA and PKD1 can both phosphorylate HSP20 on serine 16 but that PKA is the most dominant.

Disruption of the pro-oncogenic c-RAF-PDE8A complex represents a differentiated approach to treating KRAS-c-RAF dependent PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is considered the third leading cause of cancer mortality in the western world, offering advanced stage patients with few viable treatment options. Consequently, there remains an urgent unmet need to develop novel therapeutic strategies that can effectively inhibit pro-oncogenic molecular targets underpinning PDACs pathogenesis and progression. One such target is c-RAF, a downstream effector of RAS that is considered essential for the oncogenic growth and survival of mutant RAS-driven cancers (including KRASMT PDAC). Herein, we demonstrate how a novel cell-penetrating peptide disruptor (DRx-170) of the c-RAF-PDE8A protein-protein interaction (PPI) represents a differentiated approach to exploiting the c-RAF-cAMP/PKA signaling axes and treating KRAS-c-RAF dependent PDAC. Through disrupting the c-RAF-PDE8A protein complex, DRx-170 promotes the inactivation of c-RAF through an allosteric mechanism, dependent upon inactivating PKA phosphorylation. DRx-170 inhibits cell proliferation, adhesion and migration of a KRASMT PDAC cell line (PANC1), independent of ERK1/2 activity. Moreover, combining DRx-170 with afatinib significantly enhances PANC1 growth inhibition in both 2D and 3D cellular models. DRx-170 sensitivity appears to correlate with c-RAF dependency. This proof-of-concept study supports the development of DRx-170 as a novel and differentiated strategy for targeting c-RAF activity in KRAS-c-RAF dependent PDAC.

Beta-amyloid interacts with and activates the long-form phosphodiesterase PDE4D5 in neuronal cells to reduce cAMP availability.

Inhibition of the cyclic-AMP degrading enzyme phosphodiesterase type 4 (PDE4) in the brains of animal models is protective in Alzheimer's disease (AD). We show for the first time that enzymes from the subfamily PDE4D not only colocalize with beta-amyloid (Aß) plaques in a mouse model of AD but that Aß directly associates with the catalytic machinery of the enzyme. Peptide mapping suggests that PDE4D is the preferential PDE4 subfamily for Aß as it possesses a unique binding site. Intriguingly, exogenous addition of Aß to cells overexpressing the PDE4D5 longform caused PDE4 activation and a decrease in cAMP. We suggest a novel mechanism where PDE4 longforms can be activated by Aß, resulting in the attenuation of cAMP signalling to promote loss of cognitive function in AD.

Protein kinase D in the hypertrophy pathway.

Chronic neurohormonal stimulation can have direct adverse effects on the structure and function of the heart. Heart failure develops and progresses as a result of the deleterious changes. It is well established that phosphorylation of class II HDAC5 (histone deacetylase 5) is an important event in the transcriptional regulation of cardiac gene reprogramming that results in the hypertrophic growth response. To date, experimentation on phosphorylation-mediated translocation of HDAC5 has focused on the regulatory properties of PKD (protein kinase D) within intact cells. With regard to the potential role of PKD in myocardium, recent observations raise the possibility that PKD-mediated myocardial regulatory mechanisms may represent promising therapeutic avenues for the treatment of heart failure. The present review summarizes the most recent and important insights into the role of PKD in hypertrophic signalling pathways.

Material-driven fibronectin assembly rescues matrix defects due to mutations in collagen IV in fibroblasts.

Basement membranes (BMs) are specialised extracellular matrices that provide structural support to tissues as well as influence cell behaviour and signalling. Mutations in COL4A1/COL4A2, a major BM component, cause a familial form of eye, kidney and cerebrovascular disease, including stroke, while common variants in these genes are a risk factor for intracerebral haemorrhage in the general population. These phenotypes are associated with matrix defects, due to mutant protein incorporation in the BM and/or its absence by endoplasmic reticulum (ER) retention. However, the effects of these mutations on matrix stiffness, the contribution of the matrix to the disease mechanism(s) and its effects on the biology of cells harbouring a collagen IV mutation remain poorly understood. To shed light on this, we employed synthetic polymer biointerfaces, poly(ethyl acrylate) (PEA) and poly(methyl acrylate) (PMA) coated with ECM proteins laminin or fibronectin (FN), to generate controlled microenvironments and investigate their effects on the cellular phenotype of primary fibroblasts harbouring a COL4A2+/G702D mutation. FN nanonetworks assembled on PEA induced increased deposition and assembly of collagen IV in COL4A2+/G702D cells, which was associated with reduced ER size and enhanced levels of protein chaperones such as BIP, suggesting increased protein folding capacity of the cell. FN nanonetworks on PEA also partially rescued the reduced stiffness of the deposited matrix and cells, and enhanced cell adhesion through increased actin-myosin contractility, effectively rescuing some of the cellular phenotypes associated with COL4A1/4A2 mutations. The mechanism by which FN nanonetworks enhanced the cell phenotype involved integrin ß1-mediated signalling. Collectively, these results suggest that biomaterials and enhanced integrin signalling via assembled FN are able to shape the matrix and cellular phenotype of the COL4A2+/G702D mutation in patient-derived cells.

Basement membrane collagens and disease mechanisms.

Basement membranes (BMs) are specialised extracellular matrix (ECM) structures and collagens are a key component required for BM function. While collagen IV is the major BM collagen, collagens VI, VII, XV, XVII and XVIII are also present. Mutations in these collagens cause rare multi-systemic diseases but these collagens have also been associated with major common diseases including stroke. Developing treatments for these conditions will require a collective effort to increase our fundamental understanding of the biology of these collagens and the mechanisms by which mutations therein cause disease. Novel insights into pathomolecular disease mechanisms and cellular responses to these mutations has been exploited to develop proof-of-concept treatment strategies in animal models. Combined, these studies have also highlighted the complexity of the disease mechanisms and the need to obtain a more complete understanding of these mechanisms. The identification of pathomolecular mechanisms of collagen mutations shared between different disorders represent an attractive prospect for treatments that may be effective across phenotypically distinct disorders.

Transplantation of Gene-Edited Hepatocyte-like Cells Modestly Improves Survival of Arginase-1-Deficient Mice.

Progress in gene editing research has been accelerated by utilizing engineered nucleases in combination with induced pluripotent stem cell (iPSC) technology. Here, we report transcription activator-like effector nuclease (TALEN)-mediated reincorporation of Arg1 exons 7 and 8 in iPSCs derived from arginase-1-deficient mice possessing Arg1Δ alleles lacking these terminal exons. The edited cells could be induced to differentiate into hepatocyte-like cells (iHLCs) in vitro and were subsequently used for transplantation into our previously described (Sin et al., PLoS ONE 2013) tamoxifen-inducible Arg1-Cre arginase-1-deficient mouse model. While successful gene-targeted repair was achieved in iPSCs containing Arg1Δ alleles, only minimal restoration of urea cycle function could be observed in the iHLC-transplanted mice compared to control mice, and survival in this lethal model was extended by up to a week in some mice. The partially rescued phenotype may be due to inadequate regenerative capacity of arginase-1-expressing cells in the correct metabolic zones. Technical hurdles exist and will need to be overcome for gene-edited iPSC to iHLC rescue of arginase-1 deficiency, a rare urea cycle disorder.

Proof-of-Concept Gene Editing for the Murine Model of Inducible Arginase-1 Deficiency.

Arginase-1 deficiency in humans is a rare genetic disorder of metabolism resulting from a loss of arginase-1, leading to impaired ureagenesis, hyperargininemia and neurological deficits. Previously, we generated a tamoxifen-inducible arginase-1 deficient mouse model harboring a deletion of Arg1 exons 7 and 8 that leads to similar biochemical defects, along with a wasting phenotype and death within two weeks. Here, we report a strategy utilizing the Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system in conjunction with piggyBac technology to target and reincorporate exons 7 and 8 at the specific Arg1 locus in attempts to restore the function of arginase-1 in induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (iHLCs) and macrophages in vitro. While successful gene targeted repair was achieved, minimal urea cycle function was observed in the targeted iHLCs compared to adult hepatocytes likely due to inadequate maturation of the cells. On the other hand, iPSC-derived macrophages expressed substantial amounts of "repaired" arginase. Our studies provide proof-of-concept for gene-editing at the Arg1 locus and highlight the challenges that lie ahead to restore sufficient liver-based urea cycle function in patients with urea cycle disorders.

Liver-specific knockout of arginase-1 leads to a profound phenotype similar to inducible whole body arginase-1 deficiency.

Arginase-1 (Arg1) converts arginine to urea and ornithine in the distal step of the urea cycle in liver. We previously generated a tamoxifen-inducible Arg1 deficient mouse model (Arg1-Cre) that disrupts Arg1 expression throughout the whole body and leads to lethality ≈ 2 weeks after gene disruption. Here, we evaluate if liver-selective Arg1 loss is sufficient to recapitulate the phenotype observed in global Arg1 knockout mice, as well as to gauge the effectiveness of gene delivery or hepatocyte transplantation to rescue the phenotype. Liver-selective Arg1 deletion was induced by using an adeno-associated viral (AAV)-thyroxine binding globulin (TBG) promoter-Cre recombinase vector administered to Arg1 "floxed" mice; Arg1fl/fl ). An AAV vector expressing an Arg1-enhanced green fluorescent protein (Arg1-eGFP) transgene was used for gene delivery, while intrasplenic injection of wild-type (WT) C57BL/6 hepatocytes after partial hepatectomy was used for cell delivery to "rescue" tamoxifen-treated Arg1-Cre mice. The results indicate that liver-selective loss of Arg1 (> 90% deficient) leads to a phenotype resembling the whole body knockout of Arg1 with lethality ≈ 3 weeks after Cre-induced gene disruption. Delivery of Arg1-eGFP AAV rescues more than half of Arg1 global knockout male mice (survival > 4 months) but a significant proportion still succumb to the enzyme deficiency even though liver expression and enzyme activity of the fusion protein reach levels observed in WT animals. Significant Arg1 enzyme activity from engrafted WT hepatocytes into knockout livers can be achieved but not sufficient for rescuing the lethal phenotype. This raises a conundrum relating to liver-specific expression of Arg1. On the one hand, loss of expression in this organ appears to be both necessary and sufficient to explain the lethal phenotype of the genetic disorder in mice. On the other hand, gene and cell-directed therapies suggest that rescue of extra-hepatic Arg1 expression may also be necessary for disease correction. Further studies are needed in order to illuminate the detailed mechanisms for pathogenesis of Arg1-deficiency.

Arginase-1 deficiency.

Arginase-1 (ARG1) deficiency is a rare autosomal recessive disorder that affects the liver-based urea cycle, leading to impaired ureagenesis. This genetic disorder is caused by 40+ mutations found fairly uniformly spread throughout the ARG1 gene, resulting in partial or complete loss of enzyme function, which catalyzes the hydrolysis of arginine to ornithine and urea. ARG1-deficient patients exhibit hyperargininemia with spastic paraparesis, progressive neurological and intellectual impairment, persistent growth retardation, and infrequent episodes of hyperammonemia, a clinical pattern that differs strikingly from other urea cycle disorders. This review briefly highlights the current understanding of the etiology and pathophysiology of ARG1 deficiency derived from clinical case reports and therapeutic strategies stretching over several decades and reports on several exciting new developments regarding the pathophysiology of the disorder using ARG1 global and inducible knockout mouse models. Gene transfer studies in these mice are revealing potential therapeutic options that can be exploited in the future. However, caution is advised in extrapolating results since the lethal disease phenotype in mice is much more severe than in humans indicating that the mouse models may not precisely recapitulate human disease etiology. Finally, some of the functions and implications of ARG1 in non-urea cycle activities are considered. Lingering questions and future areas to be addressed relating to the clinical manifestations of ARG1 deficiency in liver and brain are also presented. Hopefully, this review will spark invigorated research efforts that lead to treatments with better clinical outcomes.

Strategies to rescue the consequences of inducible arginase-1 deficiency in mice.

Arginase-1 catalyzes the conversion of arginine to ornithine and urea, which is the final step of the urea cycle used to remove excess ammonia from the body. Arginase-1 deficiency leads to hyperargininemia in mice and man with severe lethal consequences in the former and progressive neurological impairment to varying degrees in the latter. In a tamoxifen-induced arginase-1 deficient mouse model, mice succumb to the enzyme deficiency within 2 weeks after inducing the knockout and retain <2 % enzyme in the liver. Standard clinical care regimens for arginase-1 deficiency (low-protein diet, the nitrogen-scavenging drug sodium phenylbutyrate, ornithine supplementation) either failed to extend lifespan (ornithine) or only minimally prolonged lifespan (maximum 8 days with low-protein diet and drug). A conditional, tamoxifen-inducible arginase-1 transgenic mouse strain expressing the enzyme from the Rosa26 locus modestly extended lifespan of neonatal mice, but not that of 4-week old mice, when crossed to the inducible arginase-1 knockout mouse strain. Delivery of an arginase-1/enhanced green fluorescent fusion construct by adeno-associated viral delivery (rh10 serotype with a strong cytomegalovirus-chicken ß-actin hybrid promoter) rescued about 30% of male mice with lifespan prolongation to at least 6 months, extensive hepatic expression and restoration of significant enzyme activity in liver. In contrast, a vector of the AAV8 serotype driven by the thyroxine-binding globulin promoter led to weaker liver expression and did not rescue arginase-1 deficient mice to any great extent. Since the induced arginase-1 deficient mouse model displays a much more severe phenotype when compared to human arginase-1 deficiency, these studies reveal that it may be feasible with gene therapy strategies to correct the various manifestations of the disorder and they provide optimism for future clinical studies.

Specific interactions between Epac1, ß-arrestin2 and PDE4D5 regulate ß-adrenergic receptor subtype differential effects on cardiac hypertrophic signaling.

ß1 and ß2 adrenergic receptors (ßARs) are highly homologous but fulfill distinct physiological and pathophysiological roles. Here we show that both ßAR subtypes activate the cAMP-binding protein Epac1, but they differentially affect its signaling. The distinct effects of ßARs on Epac1 downstream effectors, the small G proteins Rap1 and H-Ras, involve different modes of interaction of Epac1 with the scaffolding protein ß-arrestin2 and the cAMP-specific phosphodiesterase (PDE) variant PDE4D5. We found that ß-arrestin2 acts as a scaffold for Epac1 and is necessary for Epac1 coupling to H-Ras. Accordingly, knockdown of ß-arrestin2 prevented Epac1-induced histone deacetylase 4 (HDAC4) nuclear export and cardiac myocyte hypertrophy upon ß1AR activation. Moreover, Epac1 competed with PDE4D5 for interaction with ß-arrestin2 following ß2AR activation. Dissociation of the PDE4D5-ß-arrestin2 complex allowed the recruitment of Epac1 to ß2AR and induced a switch from ß2AR non-hypertrophic signaling to a ß1AR-like pro-hypertrophic signaling cascade. These findings have implications for understanding the molecular basis of cardiac myocyte remodeling and other cellular processes in which ßAR subtypes exert opposing effects.

Inducible arginase 1 deficiency in mice leads to hyperargininemia and altered amino acid metabolism.

Arginase deficiency is a rare autosomal recessive disorder resulting from a loss of the liver arginase isoform, arginase 1 (ARG1), which is the final step in the urea cycle for detoxifying ammonia. ARG1 deficiency leads to hyperargininemia, characterized by progressive neurological impairment, persistent growth retardation and infrequent episodes of hyperammonemia. Using the Cre/loxP-directed conditional gene knockout system, we generated an inducible Arg1-deficient mouse model by crossing "floxed" Arg1 mice with CreER(T2) mice. The resulting mice (Arg-Cre) die about two weeks after tamoxifen administration regardless of the starting age of inducing the knockout. These treated mice were nearly devoid of Arg1 mRNA, protein and liver arginase activity, and exhibited symptoms of hyperammonemia. Plasma amino acid analysis revealed pronounced hyperargininemia and significant alterations in amino acid and guanidino compound metabolism, including increased citrulline and guanidinoacetic acid. Despite no alteration in ornithine levels, concentrations of other amino acids such as proline and the branched-chain amino acids were reduced. In summary, we have generated and characterized an inducible Arg1-deficient mouse model exhibiting several pathologic manifestations of hyperargininemia. This model should prove useful for exploring potential treatment options of ARG1 deficiency.