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Peter H Sugden

Publications and source records attributed to Peter H Sugden.

At least 19 recordsLinked to original sources
PubMed2006

Cardiac myocyte gene expression profiling during H2O2-induced apoptosis.

High levels of oxidative stress promote cardiac myocyte death, though lower levels are potentially cytoprotective/anabolic. We examined the changes in gene expression in rat neonatal cardiac myocytes exposed to apoptotic (0.2 mM) or nontoxic (0.04 mM) concentrations of H2O2 (2, 4, or 24 h) using Affymetrix microarrays. Using U34B arrays, we identified a ubiquitously expressed, novel H2O2-responsive gene [putative peroxide-inducible transcript 1 (Perit1)], which generates two alternatively spliced transcripts. Using 230 2.0 arrays, H2O2 (0.04 mM) promoted significant changes in expression of only 32 genes, all of which were seen with 0.2 mM H2O2. We failed to detect any increase in the rate of protein synthesis in cardiac myocytes exposed to <0.1 mM H2O2, further suggesting that global, low concentrations of H2O2 are not anabolic in this system. H2O2 (0.2 mM) promoted significant (P < 0.05, >1.75-fold) changes in expression of 649 mRNAs and 187 RNAs corresponding to no established gene. Of the mRNAs, 114 encoded transcriptional regulators including Krüppel-like factors (Klfs). Quantitative PCR independently verified the changes in Klf expression. Thus, H2O2-induced cardiac myocyte apoptosis is associated with dynamic changes in gene expression. The expression of these genes and their protein products potentially influences the progression of the apoptotic response.

Animals↗
PubMed2006

Using U0126 to dissect the role of the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade in the regulation of gene expression by endothelin-1 in cardiac myocytes.

The hypertrophic agonist endothelin-1 rapidly but transiently activates the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade (and other signalling pathways) in cardiac myocytes, but the events linking this to hypertrophy are not understood. Using Affymetrix rat U34A microarrays, we identified the short-term (2-4 h) changes in gene expression induced in neonatal myocytes by endothelin-1 alone or in combination with the ERK1/2 cascade inhibitor, U0126. Expression of 15 genes was significantly changed by U0126 alone, and expression of an additional 78 genes was significantly changed by endothelin-1. Of the genes upregulated by U0126, four are classically induced through the aryl hydrocarbon receptor (AhR) by dioxins suggesting that U0126 activates the xenobiotic response element in cardiac myocytes potentially independently of effects on ERK1/2 signalling. The 78 genes showing altered expression with endothelin-1 formed five clusters: (i) three clusters showing upregulation by endothelin-1 according to time course (4 h > 2 h; 2 h > 4 h; 2 h approximately 4 h) with at least partial inhibition by U0126; (ii) a cluster of 11 genes upregulated by endothelin-1 but unaffected by U0126 suggesting regulation through signalling pathways other than ERK1/2; (iii) a cluster of six genes downregulated by endothelin-1 with attenuation by U0126. Thus, U0126 apparently activates the AhR in cardiac myocytes (which must be taken into account in protracted studies), but careful analysis allows identification of genes potentially regulated acutely via the ERK1/2 cascade. Our data suggest that the majority of changes in gene expression induced by endothelin-1 are mediated by the ERK1/2 cascade.

Animals↗
PubMed2005

Regulation of protein kinase C delta by phorbol ester, endothelin-1, and platelet-derived growth factor in cardiac myocytes.

Protein kinase C (PKC) delta is regulated allosterically by phosphatidylserine and diacylglycerol (which promote its translocation to the membrane) and by phosphorylation of Ser/Thr and Tyr residues. Although phosphorylation on Thr-505/Ser-643/Ser-662 may simply "prime" PKCdelta for activation, it could be regulatory. We examined the regulation of PKCdelta in cardiac myocytes by endothelin-1 (Gq protein-coupled receptor agonist) and platelet-derived growth factor (receptor tyrosine kinase agonist) in comparison with phorbol 12-myristate 13-acetate (PMA). All increased phosphorylation of PKCdelta(Thr-505/Ser-643) and of Tyr residues, although to differing extents. De novo phosphorylation occurred mainly after translocation of PKCdelta to the particulate fraction, and phosphorylations of Thr-505/Ser-643 versus Tyr residues were essentially independent events. Following chromatographic separation of the PKCdelta subspecies, activities were correlated with immunoreactivity profiles of total and phosphorylated forms. In unstimulated cells, approximately 25% of PKCdelta lacked phosphorylation of Thr-505/Ser-643 and displayed minimal activity (assayed in the presence of phosphatidylserine/PMA following chromatography). Endothelin-1 or PMA (10 min) promoted Thr-505/Ser-643 phosphorylation of this pool, and this was associated with an increase in total recoverable PKCdelta activity. Meanwhile, in cells exposed to endothelin-1 or PMA, the overall pool of PKCdelta translocated rapidly (30 s) to the particulate fraction and was phosphorylated on Tyr residues. This was associated with an increase in lipid-independent activity (i.e. the phosphatidylserine/PMA requirement disappeared). For endothelin-1, Tyr phosphorylation of PKCdelta and the increase in phosphatidylserine/PMA-independent activity persisted after PKCdelta retrotranslocated to the soluble fraction. We concluded that, with this physiological agonist, PKCdelta becomes activated in the particulate fraction but retains activity following its retrotranslocation, presumably to phosphorylate substrates elsewhere.

Animals↗
PubMed2005

Effects of oxidative stress on the cardiac myocyte proteome: modifications to peroxiredoxins and small heat shock proteins.

Endogenous oxidative stress is a likely cause of cardiac myocyte death in vivo. We examined the early (0-2 h) changes in the proteome of isolated cardiac myocytes from neonatal rats exposed to H2O2 (0.1 mM), focussing on proteins with apparent molecular masses of between 20 and 30 kDa. Proteins were separated by two-dimensional gel electrophoresis (2DGE), located by silver-staining and identified by mass spectrometry. Incorporation of [35S]methionine or 32Pi was also studied. For selected proteins, transcript abundance was examined by reverse transcriptase-polymerase chain reaction. Of the 38 protein spots in the region, 23 were identified. Two families showed changes in 2DGE migration or abundance with H2O2 treatment: the peroxiredoxins and two small heat shock protein (Hsp) family members: heat shock 27 kDa protein 1 (Hsp25) and alphaB-crystallin. Peroxiredoxins shifted to lower pI values and this was probably attributable to 'over-oxidation' of active site Cys-residues. Hsp25 also shifted to lower pI values but this was attributable to phosphorylation. alphaB-crystallin migration was unchanged but its abundance decreased. Transcripts encoding peroxiredoxins 2 and 5 increased significantly. In addition, 10 further proteins were identified. For two (glutathione S-transferase pi, translationally-controlled tumour protein), we could not find any previous references indicating their occurrence in cardiac myocytes. We conclude that exposure of cardiac myocytes to oxidative stress causes post-translational modification in two protein families involved in cytoprotection. These changes may be potentially useful diagnostically. In the short term, oxidative stress causes few detectable changes in global protein abundance as assessed by silver-staining.

Amino Acid Sequence↗
PubMed2005

Regulation of gene and protein expression in cardiac myocyte hypertrophy and apoptosis.

Considerable efforts have been expended in elucidating the inter-cellular and intra-cellular signaling pathways which elicit cardiac myocyte hypertrophy or apoptosis, and in identifying the changes which are associated with the end-stage of the response. The challenge now is to link the two. Although some of the signaling effects will be the acute modulation of existing protein function, long-term effects which bring about and maintain the hypertrophic state or which culminate in cell death are mediated at the level of gene and protein expression. With the advances in micro-array technology and genome sequencing, it is now possible to obtain a picture of the global gene expression profile in myocytes or in whole heart which dictates the proteins which could be made. This is not the final picture since additional regulation at the level of translation modulates the relative proportions of each protein that can be made from the transcriptome. Even here, further regulation of protein stability and turnover means that ultimately it is still necessary to examine the proteome to determine what may cause the functional changes in a cell. Thus, in order to gain a full picture of events which regulate the response and gain some insight into possible points of intervention for therapy, it is necessary to examine gene expression, mRNA translation and protein expression in concert.

Animals↗
PubMed2005

Peptide growth factors signal differentially through protein kinase C to extracellular signal-regulated kinases in neonatal cardiomyocytes.

The extracellular signal-regulated kinases 1/2 (ERK1/2) are activated in cardiomyocytes by Gq protein-coupled receptors and are associated with induction of hypertrophy. Here, we demonstrate that, in primary cardiomyocyte cultures, ERK1/2 were also significantly activated by platelet-derived growth factor (PDGF), epidermal growth factor (EGF) or fibroblast growth factor (FGF), but insulin, insulin-like growth factor 1 (IGF-1) and nerve growth factor (NGF) had relatively minor effects. PDGF, EGF or FGF increased cardiomyocyte size via ERK1/2, whereas insulin, IGF-1 or NGF had no effect suggesting minimum thresholds/durations of ERK1/2 signaling are required for the morphological changes associated with hypertrophy. Peptide growth factors are widely accepted to activate phospholipase C gamma1 (PLCgamma1) and protein kinase C (PKC). In cardiomyocytes, only PDGF stimulated tyrosine phosphorylation of PLCgamma1 and nPKCdelta. Furthermore, activation of ERK1/2 by PDGF, but not EGF, required PKC activity. In contrast, EGF substantially increased Ras.GTP with rapid activation of c-Raf, whereas stimulation of Ras.GTP loading by PDGF was minimal and activation of c-Raf was delayed. Our data provide clear evidence for differential coupling of PDGF and EGF receptors to the ERK1/2 cascade, and indicate that a minimum threshold/duration of ERK1/2 signaling is required for the development of cardiomyocyte hypertrophy.

Animals↗
PubMed2005

Signaling properties and expression in normal and tumor tissues of two phospholipase C epsilon splice variants.

Phospholipase Cepsilon (PLCepsilon) is a novel member of phosphoinositide-specific phospholipase C enzymes with a unique regulatory link to Ras GTP-ases. In the present studies, we establish existence of two splice variants (PLCepsilon1a and PLCepsilon1b) derived from human PLCepsilon1 gene. When expressed in COS or HEK293 cells, PLCepsilon1a and PLCepsilon1b have similar potential to be stimulated by diverse signaling pathways via tyrosine kinase and G-protein coupled receptors and share the ability to function as an effector of Ras. The expression pattern shows broader mRNA expression of PLCepsilon1a in normal tissues; furthermore, in most cell lines expressing PLCepsilon, PLCepsilon1a is the only splice variant present. Analysis of normal/tumor matched pairs derived from colon and rectum demonstrates greatly reduced expression levels in tumor tissues. Further studies in a colorectal tumor cell line lacking PLCepsilon show restoration of transcription of PLCepsilon1a and PLCepsilon1b by demethylating agent 5-aza-2'-deoxycytidine, suggesting epigenetic silencing through hypermethylation. In addition, expression of exogenous PLCepsilon in this cell line demonstrates inhibitory effects of PLCepsilon on cell viability and proliferation. Taken together, our findings suggest that regulatory mechanisms controlling expression of PLCepsilon, broadened by diversity introduced by splice variants, could play important role in PLCepsilon regulation in normal and tumor cells.

Alternative Splicing↗
PubMed2005

Endothelin signalling in the cardiac myocyte and its pathophysiological relevance.

Endothelin A (ET(A)) transmembrane receptors predominate in rat cardiac myocytes. These are G protein-coupled receptors whose actions are mediated by the G(q) heterotrimeric G proteins. Through these, ET-1 binding to ET(A)-receptors stimulates the hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate to diacylglycerol and inositol 1,4,5-trisphosphate. Diacylglycerol remains in the membrane whereas inositol 1,4,5-trisphosphate is soluble (though its importance in the cardiac myocyte is still debated). Isoforms of the phospholipid-dependent protein kinase, protein kinase C (PKC), are intracellular receptors for diacylglycerol. Cytoplasmic nPKCdelta and nPKCepsilon detect increases in membrane diacylglycerols and translocate to the membrane. This brings about PKC activation, though modifications additional to binding to phospholipids and diacylglycerol are involved. The next event (probably associated with PKC activation) is the activation of the membrane-bound small G protein Ras by exchange of GTP for GDP. Ras.GTP loading translocates Raf family mitogen-activated protein kinase (MAPK) kinase kinases to the membrane, initiates the activation of Raf, and thus activates the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade. Over longer times, two analogous protein kinase cascades, the c-Jun N-terminal kinase and p38-mitogen-activated protein kinase cascades, become activated. As the signals originating from the ET(A) receptor are transmitted through these protein kinase pathways, other signalling molecules become phosphorylated, thus changing their biological activities. For example, ET-1 increases the expression of the c-jun transcription factor gene, and increases abundance and phosphorylation of c-Jun protein. These changes in c-Jun expression and phosphorylation are likely to be important in the regulation of gene transcription.

Animals↗
PubMed2004

Endothelin-1 promotes phosphorylation of CREB transcription factor in primary cultures of neonatal rat cardiac myocytes: implications for the regulation of c-jun expression.

Cardiac myocyte hypertrophy is associated with an increase in expression of immediate early genes (e.g. c-jun) via activation of pre-existing transcription factors. The activity of CREB transcription factor is regulated through phosphorylation of Ser-133 by one of several protein kinases (e.g. protein kinase A (PKA), p90 ribosomal S6 kinases (RSKs) and the related kinase, MSK1). A cell-permeable form of cAMP, hypertrophic agonists (endothelin-1 (ET-1), phenylephrine (PE)) and hyperosmotic shock all promoted phosphorylation of CREB(Ser-133) in rat neonatal cardiac myocytes. The response to endothelin-1 required the extracellular signal-regulated kinase cascade which stimulates both RSKs and MSK1. Phosphorylation of CREB(Ser-133) in response to ET-1 was not associated with any increase in DNA binding to a consensus cAMP-response element (CRE). The rat c-jun promoter contains elements which may bind either c-Jun/ATF2 or CREB/ATF1 dimers. Using extracts from rat cardiac myocytes, we identified at least two complexes which bind to the most proximal of these elements, one of which contained CREB and the other c-Jun. Thus, phosphorylation and activation of CREB in cardiac myocytes may be effected by a range of different stimuli to influence the expression of immediate early genes such as c-jun.

Animals↗
PubMed2004

Phenylephrine and endothelin-1 upregulate connective tissue growth factor in neonatal rat cardiac myocytes.

Cardiac hypertrophy is associated with hypertrophic growth of cardiac myocytes and increased fibrosis. Much is known of the stimuli which promote myocyte hypertrophy and the changes associated with the response, but the links between the two are largely unknown. Using subtractive hybridization, we identified three genes which are acutely (<1 h) upregulated in neonatal rat ventricular myocytes exposed to the alpha-adrenergic agonist, phenylephrine. One represented connective tissue growth factor (CTGF) which is implicated in fibrosis and promotes hypertrophy in other cells. We further examined the expression of CTGF mRNA and protein in cardiac myocytes using quantitative PCR and immunoblotting, confirming that phenylephrine increased CTGF mRNA (maximal within 1 h) and protein (increased over 4 - 24 h). Endothelin-1 promoted a greater, though transient, increase in CTGF mRNA, but the increase in CTGF protein was sustained over 8 h. Neither agonist increased CTGF mRNA in cardiac non-myocytes. By increasing the expression of CTGF in cardiac myocytes, hypertrophic agonists such as phenylephrine and endothelin-1 may promote fibrosis. CTGF may also propagate the hypertrophic response initiated by these agonists.

Animals↗
PubMed2004

Signaling through the extracellular signal-regulated kinase 1/2 cascade in cardiac myocytes.

The extracellular signal-regulated kinases 1/2 (ERK1/2) are particularly implicated in the growth response of cardiac myocytes. In these cells, the ERK1/2 pathway is potently activated by Gq protein-coupled receptor agonists (such as endothelin-1 or alpha-adrenergic agonists), which activate protein kinase C isoforms. Here, we review the mechanisms associated with the activation of the ERK1/2 pathway by these agonists with particular emphasis on signal integration into the pathway. Signaling to the nucleus and the regulation of transcription factor activity associated with ERK1/2 activation in cardiac myocytes are also discussed.

Animals↗
PubMed2003

Ras, Akt, and mechanotransduction in the cardiac myocyte.

The Ras subfamily of 21-kDa ("small") guanine nucleotide binding proteins [which includes Ha-Ras, Ki(A)-Ras, Ki(B)-Ras, and N-Ras] is universally important in regulating intracellular signaling events in mammalian cells and controls their growth, proliferation, senescence, differentiation, and survival. These Ras isoforms act as membrane-associated biological switches that transduce signals from transmembrane receptors, thus potentially activating a variety of downstream signaling proteins. These include ultimately two Ser/Thr protein kinase families, the extracellular signal-regulated kinases 1/2 (ERK1/2) and Akt (or protein kinase B). Activation of ERK1/2 has been associated with cardiac myocyte hypertrophy (ie, increased cell size and myofibrillogenesis, with concurrent transcriptional changes to a fetal pattern of gene expression), whereas activation of Akt is associated with the increased protein accretion in hypertrophy. Both ERK1/2 and Akt may promote myocyte survival. In the intact heart in vivo and in primary cultures of cardiac myocytes, mechanical strain induces hypertrophy, a process known as mechanotransduction, which may involve Ras, ERK1/2, and Akt. In this study, general and cardiospecific aspects of the regulation of Ras and Akt will be described. The various mechanisms through which mechanical strain might initiate Ras- or Akt-dependent signaling will be discussed. The overall conclusion is that although an involvement of Ras and Akt in mechanotransduction is likely, more work (particularly focusing on mechanoreception) needs to be undertaken before it is unequivocally established.

Animals↗
PubMed2003

An overview of endothelin signaling in the cardiac myocyte.

Three endothelin (ET) isopeptides have been identified: ET-1, ET-2 and ET-3. These have two well-established gross effects on the cardiac myocyte. They affect the contractile properties and they stimulate myocyte growth and myofibrillogenesis. There may be other effects that are less fully characterized (e.g. increased resistance apoptosis). The changes in myocyte biology are brought about by modulation of intracellular signaling pathways. ET-1 binds to the ET(A) receptor on the cell surface and stimulates hydrolysis of phosphatidylinositol 4', 5'-bisphosphate to diacylglycerol and inositol 1', 4', 5'-trisphosphate. Diacylglycerol remains in the plane of the membrane and this causes translocation of the delta- and epsilon-isoforms of protein kinase C (PKC) to that compartment, an event thought to be indicative of PKC activation. The next events (probably associated with PKC activation) are the activation of the small G-protein Ras and of the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade. Over a longer time course, two protein kinase cascades related to the ERK1/2 cascade, the c-Jun N-terminal kinase and p38-mitogen-activated protein kinase (p38-mitogen) cascades, also become activated. As the signals originating from the ET(A) receptor are transmitted through these protein kinase pathways, other signaling molecules become phosphorylated, thus changing their biological activity. Such molecules include nuclear transcription factors (e.g. GATA-4, c-Jun), protein kinases (e.g. 90-kDa ribosomal protein S6 kinase, MAPK-activated protein kinase 2), and ion exchangers/channels (e.g. the Na(+)/H(+) exchanger 1). These changes are responsible for the overall biological effects of ET isopeptides on the myocyte.

Animals↗
PubMed2002

Up-regulation of c-jun mRNA in cardiac myocytes requires the extracellular signal-regulated kinase cascade, but c-Jun N-terminal kinases are required for efficient up-regulation of c-Jun protein.

Cardiac hypertrophy, an important adaptational response, is associated with up-regulation of the immediate early gene, c- jun, which encodes the c-Jun transcription factor. c-Jun may feed back to up-regulate its own transcription and, since the c-Jun N-terminal kinase (JNK) family of mitogen-activated protein kinases (MAPKs) phosphorylate c-Jun(Ser-63/73) to increase its transactivating activity, JNKs are thought to be the principal factors involved in c- jun up-regulation. Hypertrophy in primary cultures of cardiac myocytes is induced by endothelin-1, phenylephrine or PMA, probably through activation of one or more of the MAPK family. These three agonists increased c- jun mRNA with the rank order of potency of PMA approximately endothelin-1>phenylephrine. Up-regulation of c- jun mRNA by endothelin-1 was attenuated by inhibitors of protein kinase C (GF109203X) and the extracellular signal-regulated kinase (ERK) cascade (PD98059 or U0126), but not by inhibitors of the JNK (SP600125) or p38-MAPK (SB203580) cascades. Hyperosmotic shock (0.5 M sorbitol) powerfully activates JNKs, but did not increase c- jun mRNA. These data suggest that ERKs, rather than JNKs, are required for c- jun up-regulation. However, endothelin-1 and phenylephrine induced greater up-regulation of c-Jun protein than PMA and phosphorylation of c-Jun(Ser-63/73) correlated with the level of c-Jun protein. Up-regulation of c-Jun protein by endothelin-1 was attenuated by inhibitors of protein kinase C and the ERK cascade, probably correlating with a primary input of ERKs into transcription. In addition, SP600125 inhibited the phosphorylation of c-Jun(Ser-63/73), attenuated the increase in c-Jun protein induced by endothelin-1 and increased the rate of c-Jun degradation. Thus whereas ERKs are the principal MAPKs required for c- jun transcription, JNKs are necessary to stabilize c-Jun for efficient up-regulation of the protein.

Animals↗