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

The roles of HSP20 in cardiac hypertrophy

Cardiac hypertrophy often develops to compensate for hemodynamic overload and is associated with heart failure. Recent studies have revealed that overexpression and PKA-mediated phosphorylation of heat shock protein 20 (HSP20) at Ser16 can attenuate hypertrophic growth of cardiomyocytes and trigger cardioprotective functions following sustained β-adrenergic stimulation (Fan et al., 2004, 2005, 2006). However, the molecular mechanism of HSP20 induced cardioprotection remains to be fully elucidated. In order to gain insight into the protective mode of action of HSP20, I attempted to (1) investigate the spatiotemporal control of PKA-mediated phosphorylation of HSP20, as well as (2) identifying novel protein binding partners for HSP20 utilising cutting edge ProtoArray technology. Initially, I set up an in vitro hypertrophy model using sustained isoprenaline (ISO)-stimulated neonatal rat cardiomyocytes. Cell size, protein synthesis and fetal gene expression were assessed as parameters of hypertrophic growth. In the first section of my studies, members of the cAMP-specific PDE4 family were shown to form signalling complexes with HSP20, and that the PKA-mediated phosphorylation of HSP20 could be modulated by PDE4. Based on peptide array data, a cell-permeable peptide ‘bs906’ was developed to inhibit the interaction of PDE4 with HSP20. Interestingly, the disruption of the PDE4-HSP20 complex was shown to induce PKA-mediated phosphorylation of HSP20 and trigger cardioprotection against the hypertrophic response measured in neonatal cardiomyocytes upon chronic β-adrenergic stimulation. In the second part of my studies, protein kinase D1 (PKD1) was identified as one interacting partner that robustly associated with HSP20. This interaction was confirmed by biochemical and immunocytochemical means. Using similar approaches to those used for the PDE4-HSP20 interaction, a cell-permeable peptide ‘HJL09’ was generated to promote disruption of the PKD1-HSP20 complex. Experimentation using the peptide concluded that the disruption of the PKD1-HSP20 complex reduced HSP20 phosphorylation and attenuated the hypertrophic response in cultured cardiomyocytes as shown by reduced increases in cell size, protein content and actin reorganisation. In undertaking this work, I also defined a novel PKD phosphorylation site (Ser16) on HSP20 that conforms to the PKD phosphorylation motif of RxxS (also a PKA site). My biochemical data suggested that PKD1 may regulate the cardioprotective function of HSP20 via phosphorylation at Ser16. In situ proximity ligation assay (PLA) further revealed a role of HSP20 as ‘molecular escort’ in targeting the nuclear translocation of PKD1. This function, in part, may be responsible for the induction of fetal gene reexpression as selective disruption of PKD1-HSP20 complex using ‘HJL09’ hindered the nuclear influx of the complex, thereby attenuating hypertrophic signalling. In summary, these studies describe some exciting findings which provide further insight into novel signalling mechanism of cardiac hypertrophy in neonatal rat cardiomyocytes. I have shown that PKA and PKD1 exhibiting opposite functions despite sharing the phosphorylation site on HSP20. In this regard, HSP20 functions as a molecular nexus for the opposing actions of the PKA and PKD1 signalling pathways in hypertrophy, suggesting that crosstalk may occur between anti-hypertrophic and pro-hypertrophic pathways. The identification and characterisation of these complexes should help to build a better understanding of the hypertrophic signalling pathway, and may provide novel therapeutic strategies for the treatment of cardiac hypertrophy

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

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; Arg1(fl/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

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

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

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 beta-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

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 β1-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

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 β1-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

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 remodelling and cardiac hypertrophy. However, enhancement of particular aspects of cAMP/protein kinase A (PKA) signalling appears 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 signalling microdomains. Objective: How individual cAMP microdomains impact on 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 anti-hypertrophic. By real-time imaging of cAMP levels in intact myocytes and selective displacement of PKA isoforms we demonstrate that the anti-hypertrophic effect of PDE2 inhibition involves the generation of a local pool of cAMP and activation of a PKA type II subset leading to phosphorylation of the nuclear factor of activated T cells (NFAT). 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