Human atrial beta1L-adrenoceptor but not beta3-adrenoceptor activation increases force and Ca2+ current at physiological temperature

BACKGROUND AND PURPOSE It has been proposed that BRL37344, SR58611 and CGP12177 activate b3-adrenoceptors in human atrium to increase contractility and L-type Ca2+ current (ICa-L). b3-adrenoceptor agonists are potentially beneficial for the treatment of a variety of diseases but concomitant cardiostimulation would be potentially harmful. It has also been proposed that (-)-CGP12177 activates the low affinity binding site of the b1-adrenoceptor in human atrium. We therefore used BRL37344, SR58611 and (-)-CGP12177 with selective b-adrenoceptor subtype antagonists to clarify cardiostimulant b-adrenoceptor subtypes in human atrium. EXPERIMENTAL APPROACH Human right atrium was obtained from patients without heart failure undergoing coronary artery bypass or valve surgery. Cardiomyocytes were prepared to test BRL37344, SR58611 and CGP12177 effects on ICa-L. Contractile effects were determined on right atrial trabeculae. KEY RESULTS BRL37344 increased force which was antagonized by blockade of b1- and b2-adrenoceptors but not by blockade of b3-adrenoceptors with b3-adrenoceptor-selective L-748,337 (1 mM). The b3-adrenoceptor agonist SR58611 (1 nM–10 mM) did not affect atrial force. BRL37344 and SR58611 did not increase ICa-L at 37°C, but did at 24°C which was prevented by L-748,337. (-)-CGP12177 increased force and ICa-L at both 24°C and 37°C which was prevented by (-)-bupranolol (1–10 mM), but not L-748,337. CONCLUSIONS AND IMPLICATIONS We conclude that the inotropic responses to BRL37344 are mediated through b1- and b2-adrenoceptors. The inotropic and ICa-L responses to (-)-CGP12177 are mediated through the low affinity site b1L-adrenoceptor of the b1-adrenoceptor. b3-adrenoceptor-mediated increases in ICa-L are restricted to low temperatures. Human atrial b3-adrenoceptors do not change contractility and ICa-L at physiological temperature.

Ryanodine receptor phosphorylation in human heart failure

Heart failure is a complex disorder, characterized by activation of the sympathetic nervous system, leading to dysregulated Ca2+ homeostasis in cardiac myocytes and tissue remodeling. In a variety of diseases, cardiac malfunction is associated with aberrant fluxes of Ca2+ across both the surface membrane and the internal Ca2+ store, the sarcoplasmic reticulum (SR). One prominent hypothesis residues is that in heart failure, the activity of the ryanodine receptor (RyR2) Ca2+ release channel in the SR is increased due to excess phosphorylation and that this contributes to excess SR Ca2+ leak in diastole, reduced SR Ca2+ load and decreased contractility (Huke & Bers, 2008). There is controversy over which serine residues in RyR2 are hyperphosphorylated in animal models of heart failure and whether this is via the CaMKII or the PKA-linked signaling pathway. S2808, S2814 and S2030 in RyR2 have been variously claimed to be hyperphosphorylated. Our aim was to examine the degree of phosphorylation of these residues in RyR2 from failing human hearts. The use of human tissue was approved by the Human Research Ethics Committee, The Prince Charles Hospital, EC28114. Left ventricular tissue samples were obtained from an explanted heart of a patient with endstage heart failure (Emery Dreifuss Muscular Dystrophy with cardiomyopathy) and non-failing tissue was from a patient with cystic fibrosis undergoing heart-lung transplantation with no history of heart disease. SR vesicles were prepared as described by Laver et al. (1995) and examined with SDS-Page and Western Blot. Transferred proteins were probed with antibodies to detect total protein phosphorylation, phosphorylation of RyR2 serine residues S2808, S2814, S2030 and for the key proteins calsequestrin, triadin, junctin and FKBP12.6. To avoid membrane stripping artifact, each membrane was exposed to one phosphorylation-specific antibody and signal densities quantified using Bio-Rad Quantity One software. We found no distinguishable difference between failing and healthy hearts in the protein expression levels of RyR2, triadin, junctin or calsequestrin. We found an expected upregulation of total RyR2 phosphorylation in the failing heart sample, compared to a matched amount of RyR2 (quantified using densiometry) in healthy heart. Probing with antibodies detecting only the phosphorylated form of the specific RyR2 residues showed that the increase in total RyR2 phosphorylation in the failing heart was due to hyperphosphorylation of S2808 and S2814. We found that S2030 phosphorylation levels were unchanged in human heart failure. Interestingly, we found that S2030 has a basal level of phosphorylation in the healthy human heart, different from the absence of basal phosphorylation recently reported in rodent heart (Huke & Bers, 2008). Finally, preliminary results indicate that less FKBP 12.6 is associated with RyR2 in the failing heart, possibly as a consequence of PKA activation. In conclusion, residues S2808 and S2814 are hyperphosphorylated in human heart failure, presumably due to upregulation of the CaMKII and/or PKA signaling pathway as a result of chronic activation of the sympathetic nervous system. Such changes in RyR2 phosphorylation are believed to contribute to the leaky RyR2 phenotype associated with heart failure, which increases the incidence of arrhythmia and contributes to the severely impaired contractile performance of the failing heart. Huke S & Bers DM. (2008). Ryanodine receptor phosphorylation at serine 2030, 2808 and 2814 in rat cardiomyocytes. Biochemical and Biophysical Research Communications 376, 80-85. Laver DR, Roden LD, Ahern GP, Eager KR, Junankar PR & Dulhunty AF. (1995). Cytoplasmic Ca2+ inhibits the ryanodine receptor from cardiac muscle. Journal of Membrane Biology 147, 7-22. Proceedings

Edaravone containing isoindoline nitroxides for the potential treatment of cardiovascular ischaemia

A novel antioxidant for the potential treatment of ischaemia was designed by incorporating an isoindoline nitroxide into the framework of the free radical scavenger edaravone. 5-(3-Methyl-pyrazol-5-ol-1-yl)-1,1,3,3-tetramethylisoindolin-2-yloxyl 7 was prepared by N-arylation of 3-methyl-5-pyrazolone with 5-iodo-1,1,3,3-tetramethylisoindoline-2-yloxyl 8 in the presence of catalytic copper(I)iodide. Evaluation of 7, its methoxyamine derivative 10 and 5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl (CTMIO) against edaravone 1 in ischaemic rat atrial cardiomyocytes revealed significant decreases in cell death after prolonged ischaemia for each agent; however the protective effect of the novel antioxidant 7 (showing greater than 85% reduction in cell death at 100 μM) was significantly enhanced over that of edaravone 1 alone. Furthermore, the activity for 7 was found to be equal to or greater than the potent cardioprotective agent N6-cyclopentyladenosine (CPA). The methoxyamine adduct 10 and edaravone 1 showed no difference between the extent of reduction in cell death whilst CTMIO had only a modest protective effect.

Proteomics Approaches in the Identification of Molecular Signatures of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are undifferentiated, multi-potent stem cells with the ability to renew. They can differentiate into many types of terminal cells, such as osteoblasts, chondrocytes, adipocytes, myocytes, and neurons. These cells have been applied in tissue engineering as the main cell type to regenerate new tissues. However, a number of issues remain concerning the use of MSCs, such as cell surface markers, the determining factors responsible for their differentiation to terminal cells, and the mechanisms whereby growth factors stimulate MSCs. In this chapter, we will discuss how proteomic techniques have contributed to our current knowledge and how they can be used to address issues currently facing MSC research. The application of proteomics has led to the identification of a special pattern of cell surface protein expression of MSCs. The technique has also contributed to the study of a regulatory network of MSC differentiation to terminal differentiated cells, including osteocytes, chondrocytes, adipocytes, neurons, cardiomyocytes, hepatocytes, and pancreatic islet cells. It has also helped elucidate mechanisms for growth factor–stimulated differentiation of MSCs. Proteomics can, however, not reveal the accurate role of a special pathway and must therefore be combined with other approaches for this purpose. A new generation of proteomic techniques have recently been developed, which will enable a more comprehensive study of MSCs. Keywords

The roles of the preproghrelin-derived peptides - ghrelin, desacyl ghrelin and obestatin - in prostate cancer

Prostate cancer is the second most common cause of cancer related deaths in Western men. Despite the significant improvements in current treatment techniques, there is no cure for advanced metastatic, castrate-resistant disease. Early detection and prevention of progression to a castrate-resistant state may provide new strategies to improve survival. A number of growth factors have been shown to act in an autocrine/paracrine manner to modulate prostate cancer tumour growth. Our laboratory has previously shown that ghrelin and its receptors (the functional GHS-R1a and the non-functional GHS-R1b) are expressed in prostate cancer specimens and cell lines. We have shown that ghrelin increases cell proliferation in the PC3 and LNCaP prostate cancer cell lines through activation of ERK1/2, suggesting that ghrelin could regulate prostate cancer cell growth and play a role in the progression of the disease. Ghrelin is a 28 amino-acid peptide hormone, identified to be the natural ligand of the growth hormone secretagogue receptor (GHS-R1a). It is well characterised as a growth hormone releasing and as an orexigenic peptide that stimulates appetite and feeding and regulates energy expenditure and bodyweight. In addition to its orexigenic properties, ghrelin has been shown to play a regulatory role in a number of systems, including the reproductive, immune and cardiovascular systems and may play a role in a number of pathological conditions such as chronic heart failure, anorexia, cachexia, obesity, diabetes and cancer. In cancer, ghrelin and its receptor are expressed in a range of tumours and cancer cell lines and ghrelin has been demonstrated to modulate cell proliferation, apoptosis, migration and invasion in some cell types. The ghrelin gene (GHRL) encodes preproghrelin peptide, which is processed to produce three currently known functional peptides - ghrelin, desacyl ghrelin and obestatin. Prohormone convertases (PCs) have been shown to cleave the preproghrelin peptide into two primary products - the 28 amino acid peptide, ghrelin, and the remaining 117 amino acid C-terminal peptide, C-ghrelin. C-ghrelin can then be further processed to produce the 23 amino acid peptide, obestatin. Ghrelin circulates in two different forms - an octanoylated form (known as ghrelin) and a non-octanoylated form, desacyl ghrelin. The unique post-translational addition of octanoic acid to the serine 3 residue of the propeptide chain to form acylated ghrelin is catalysed by ghrelin O-acyltransferase (GOAT). This modification is necessary for binding of ghrelin to its only known functional receptor, the GHS-R1a. As desacyl ghrelin cannot bind and activate the GHS-R1a, it was initially thought to be an inactive peptide, despite the fact that it circulates at much higher levels than ghrelin. Further research has demonstrated that desacyl ghrelin is biologically active and shares some of the actions of ghrelin, as well as having some opposing and distinct roles. Interestingly, both ghrelin and desacyl ghrelin have been shown to modulate apoptosis, cell differentiation and proliferation in some cell types, and to stimulate cell proliferation through activation of ERK1/2 and PI3K/Akt pathways. The third known peptide product of the ghrelin preprohormone, obestatin, was initially thought to oppose the actions of ghrelin in appetite regulation and food intake and to mediate its effects through the G protein-coupled receptor 39 (GPR39). Subsequent research failed to reproduce the initial findings, however, and the possible anorexigenic effects of obestatin, as well as the identity of its receptor, remain unclear. Obestatin plays some important physiological roles, including roles in improving memory, the inhibition of thirst and anxiety, increased secretion of pancreatic juice, and regulation of cell proliferation, survival, apoptosis and differentiation. Preliminary studies have also shown that obestatin stimulates cell proliferation in some cell types through activation of ERK1/2, Akt and PKC pathways. Overall, however, at the commencement of this PhD project, relatively little was known regarding the functions and mechanisms of action of the preproghrelin-derived functional peptides in modulating prostate cancer cell proliferation. The roles of obestatin, and desacyl ghrelin as potential growth factors had not previously been investigated, and the potential expression and regulation of the preproghrelin processing enzymes, GOAT and prohormone convertases was unknown in prostate cancer cell lines. Therefore, the overall objectives of this study were to: 1. investigate the effects of obestatin on cell proliferation and signaling in prostate cancer cell lines 2. compare the effects of desacyl ghrelin and ghrelin on cell proliferation and signaling in prostate cancer cell lines 3. investigate whether prostate cancer cell lines possess the necessary enzymatic machinery to produce ghrelin and desacyl ghrelin and if these peptides can regulate GOAT expression Our laboratory has previously shown that ghrelin stimulates cell proliferation in the PC3 and LNCaP prostate cancer cell line through activation of the ERK1/2 pathway. In this study it has been demonstrated that treatments with either ghrelin, desacyl ghrelin or obestatin over 72 hours significantly increased cell proliferation in the PC3 prostate cancer cell line but had no significant effect in the RWPE-1 transformed normal prostate cell line. Ghrelin (1000nM) stimulated cell proliferation in the PC3 prostate cancer cell line by 31.66 6.68% (p<0.01) with the WST-1 method, and 13.55 5.68% (p<0.05) with the CyQUANT assay. Desacyl ghrelin (1000nM) increased cell proliferation in PC3 cells by 21.73 2.62% (p<0.01) (WST-1), and 15.46 7.05% (p<0.05) (CyQUANT) above untreated control. Obestatin (1000nM) induced a 28.37 7.47% (p<0.01) (WST-1) and 12.14 7.47% (p<0.05) (CyQUANT) significant increase in cell proliferation in the PC3 prostate cancer cell line. Ghrelin and desacyl ghrelin treatments stimulated Akt and ERK phosphorylation across a range of concentrations (p<0.01). Obestatin treatment significantly stimulated Akt, ERK and PKC phosphorylation (p<0.05). Through the use of specific inhibitors, the MAPK inhibitor U0126 and the Akt1/2 kinase inhibitor, it was demonstrated that ghrelin- and obestatin-induced cell proliferation in the PC3 prostate cancer cell line is mediated through activation of ERK1/2 and Akt pathways. Although desacyl ghrelin significantly stimulated Akt and ERK phosphorylation, U0126 failed to prevent desacyl ghrelin-induced cell proliferation suggesting ghrelin and desacyl ghrelin might act through different mechanisms to increase cell proliferation. Ghrelin and desacyl ghrelin have shown a proliferative effect in osteoblasts, pancreatic -cells and cardiomyocytes through activation of ERK1/2 and PI3K/Akt pathways. Here it has been shown that ghrelin and its non-acylated form exert the same function and stimulate cell proliferation in the PC3 prostate cancer cell line through activation of the Akt pathway. Ghrelin-induced proliferation was also mediated through activation of the ERK1/2 pathway, however, desacyl ghrelin seems to stimulate cell proliferation in an ERK1/2-independent manner. As desacyl ghrelin does not bind and activate GHSR1a, the only known functional ghrelin receptor, the finding that both ghrelin and desacyl ghrelin stimulate cell proliferation in the PC3 cell line suggests that these peptides could be acting through the yet unidentified alternative ghrelin receptor in this cell type. Obestatin treatment also stimulated PKC phosphorylation, however, a direct role for this pathway in stimulating cell proliferation could not be proven using available PKC pathway inhibitors, as they caused significant cell death over the extended timeframe of the cell proliferation assays. Obestatin has been shown to stimulate cell proliferation through activation of PKC isoforms in human retinal epithelial cells and in the human gastric cancer cell line KATO-III. We have demonstrated that all of the prostate-derived cell lines examined (PC3, LNCaP, DU145, 22Rv1, RWPE-1 and RWPE-2) expressed GOAT and at least one of the prohormone convertases, which are known to cleave the proghrelin peptide, PC1/3, PC2 and furin, at the mRNA level. These cells, therefore, are likely to possess the necessary machinery to cleave the preproghrelin protein and to produce the mature ghrelin and desacyl ghrelin peptides. In addition to prohormone convertases, the presence of octanoic acid is essential for acylated ghrelin production. In this study octanoic acid supplementation significantly increased cell proliferation in the PC3 prostate cancer cell line by over 20% compared to untreated controls (p<0.01), but surprisingly, not in the DU145, LNCaP or 22Rv1 prostate cancer cell lines or in the RWPE-1 and RWPE-2 prostate-derived cell lines. In addition, we demonstrated that exogenous ghrelin induced a statistically significant two-fold decrease in GOAT mRNA expression in the PC3 cell line (p<0.05), suggesting that ghrelin could pontentially downregulate its own acylation and, therefore, regulate the balance between ghrelin and desacyl ghrelin. This was not observed, however, in the DU145 and LNCaP prostate cancer cell lines. The GOAT-ghrelin system represents a direct link between ingested nutrients and regulation of ghrelin production and the ghrelin/desacyl ghrelin ratio. Regulation of ghrelin acylation is a potentially attractive and desirable tool for the development of better therapies for a number of pathological conditions where ghrelin has been shown to play a key role. The finding that desacyl ghrelin stimulates cell proliferation in the PC3 prostate cancer cell line, and responds to ghrelin in the same way, suggests that this cell line expresses an alternative ghrelin receptor. Although all the cell lines examined expressed both GHS-R1a and GHS-R1b mRNA, it remains uncertain whether these cell lines express the unidentified alternative ghrelin receptor. It is possible that the varied responses seen could be due to the expression of different ghrelin receptors in different cell lines. In addition to GOAT, prohormone convertases and octanoic acid availability may regulate the production of different peptides from the ghrelin preprohormone. The studies presented in this thesis provide significant new information regarding the roles and mechanisms of action of the preproghrelin-derived peptides, ghrelin, desacyl ghrelin and obestatin, in modulating prostate cancer cell line proliferation. A number of key questions remain to be resolved, however, including the identification of the alternative ghrelin/desacyl ghrelin receptor, the identification of the obestatin receptor, a clarification of the signaling mechanisms which mediate cell proliferation in response to obestatin treatment and a better understanding of the regulation at both the gene and post-translational levels of functional peptide generation. Further studies investigating the role of the ghrelin axis using in vivo prostate cancer models may be warranted. Until these issues are determined, the potential for the ghrelin axis, to be recognised as a novel useful target for therapy for cancer or other pathologies will be uncertain.

Mutations in cardiac T-Box Factor gene TBX20 are associated with diverse cardiac pathologies, including defects of septation and valvulogenesis and cardiomyopathy

The T-box family transcription factor gene TBX20 acts in a conserved regulatory network, guiding heart formation and patterning in diverse species. Mouse Tbx20 is expressed in cardiac progenitor cells, differentiating cardiomyocytes, and developing valvular tissue, and its deletion or RNA interference-mediated knockdown is catastrophic for heart development. TBX20 interacts physically, functionally, and genetically with other cardiac transcription factors, including NKX2-5, GATA4, and TBX5, mutations of which cause congenital heart disease (CHD). Here, we report nonsense (Q195X) and missense (I152M) germline mutations within the T-box DNA-binding domain of human TBX20 that were associated with a family history of CHD and a complex spectrum of developmental anomalies, including defects in septation, chamber growth, and valvulogenesis. Biophysical characterization of wild-type and mutant proteins indicated how the missense mutation disrupts the structure and function of the TBX20 T-box. Dilated cardiomyopathy was a feature of the TBX20 mutant phenotype in humans and mice, suggesting that mutations in developmental transcription factors can provide a sensitized template for adult-onset heart disease. Our findings are the first to link TBX20 mutations to human pathology. They provide insights into how mutation of different genes in an interactive regulatory circuit lead to diverse clinical phenotypes, with implications for diagnosis, genetic screening, and patient follow-up.