No description available.

No description available.

No / 1 Pharmacological inhibitors of protein kinase A (PKA) and protein phosphatases 1/2A were used to determine whether basal L-type Ca2+ current (ICa) observed in the absence of exogenous ß-adrenergic receptor stimulation is sustained by PKA-mediated phosphorylation. Amphotericin B was used to record whole-cell ICa in the perforated patch-clamp configuration. 2 Calyculin A and isoprenaline (both 1 ¿mol l¿1) increased basal ICa (P<0.05), whereas H-89 inhibited ICa in a concentration-dependent manner with an IC50 ~5 ¿mol l¿1. H-89 also inhibited the response to 1.0 ¿mol l¿1 isoprenaline, although relatively high concentrations (30 ¿mol l¿1) were required to achieve complete suppression of the response. 3 Double-pulse protocols were used to study the effects of 10 ¿mol l¿1 H-89 on time-dependent recovery of ICa from voltage-dependent inactivation as well as the steady-state gating of ICa. T0.5 (time for ICa to recover to 50% of the preinactivation amplitude) increased in the presence of H-89 (P<0.05) but was unaffected by calyculin A or isoprenaline. 4 Steady-state activation/inactivation properties of ICa were unaffected by 10 ¿mol l¿1 H-89 or 1 ¿mol l¿1 calyculin A, whereas isoprenaline caused a leftward shift in both curves so that V0.5 for activation and inactivation became more negative. 5 Data show that basal ICa is regulated by cAMP-PKA-mediated phosphorylation in the absence of externally applied ß-receptor agonists and that relatively high concentrations of H-89 are required to fully suppress the response to ß-adrenergic receptor stimulation, thereby limiting the value of H-89 as a useful tool in dissecting signalling pathways in intact myocytes.

Swelling-activated Cl- current (ICl,swell) is an outwardly rectifying Cl- current that influences cardiac electric activities and acts as a potential effector of mechanoelectrical feedback that antagonizes the effects of stretch-activated cation channels. Persistent activation of ICl,swell has been observed in multiple models of cardiovascular diseases. Previously we showed that angiotensin II (AngII) signaling and reactive oxygen species (ROS) produced by NADPH oxidase (NOX) are involved in the activation of ICl, swell by both beta1-integrin stretch and osmotic swelling. Because endothelin-1 (ET-1) is a potential downstream mediator of AngII and ETA receptor blockade abrogates AngII-induced ROS generation, we studied how ET-1 signaling regulates ICl,swell and the relationship between AngII and ET-1 signaling. Under isosmotic conditions, ET-1 elicited an outwardly rectifying Cl- current that was fully blocked by the highly selective ICl,swell inhibitor DCPIB and by osmotic shrinkage. Selective ETA blockade (BQ123), but not ETB blockade (BQ788), fully suppressed the ET-1-induced current. ET-1-induced ICl,swell was abolished by blockade of EGFR kinase (AG1478) and PI-3K inhibitors (LY294002 and wortmannin), which also suppress beta1-integrin stretch- and swelling-induced ICl,swell. ET-1-induced ICl,swell was abrogated by ebselen, a membrane-permeant glutathione peroxidase mimetic that dismutates H2O2 to H2O, suggesting that ROS were required intermediates in ET-1-induced activation of ICl,swell. Both NOX and mitochondria are important sources of ROS in cardiomyocytes. Blocking NOX with apocynin or mitochondrial complex I with rotenone both completely suppressed ET-1-induced ROS generation and activation of ICl,swell, indicating that ROS from both NOX and mitochondria were required to activate ICl,swell, and complete block by inhibitors of either ROS source suggests mitochondrial and NOX must act in series rather than in parallel. ICl,swell elicited by antimycin A, which stimulates superoxide production by mitochondrial complex III, was insensitive to NOX inhibitor apocynin and the NOX fusion peptide inhibitor gp91ds-tat. Activation of ICl,swell induced by diazoxide, which stimulates mitochondrial ROS production by opening mitochondrial KATP channels, was not affected by gp91ds-tat. These data suggests that mitochondrial ROS is downstream from NOX in the regulation of ICl,swell. Mitochondrial ROS production that is enhanced by NOX ROS is likely to be responsible for the activation of ICl,swell by ET-1. In order to determine the role of ERK in the proposed signaling pathway that regulates ICl,swell, we examined the effect of ERK inhibitors (PD 98059 and U0216) on the activation of ICl,swell elicited by ET-1, EGF, and H2O2. ERK inhibitors partially blocked ET-1-induced ICl,swell but fully inhibited activation of ICl,swell in response to EGF. However, ERK inhibitors did not affect ICl,swell elicited by exogenous H2O2. We also established the the relationship of ET-1 to AngII and osmotic swelling in the regulation of ET-1 ICl,swell. ETA blockade abolished ICl,swell elicited by both AngII and osmotic swelling, whereas AT1 blockade did not effect ET-1-induced ICl,swell, suggesting that ET-1 signaling is downstream from AngII and osmotic swelling. HL-1 cell is a murine atrial cell line that retain phenotypic characteristics of adult cardiomyocytes. We showed that osmotic swelling and ET-1 turned on DCPIB-sensitive outwardly rectifying Cl- current in HL-1 cells with both physiological and symmetrical Cl- gradients. The swelling-induced current was suppressed by gp91ds-tat and rotenone but insensitive to apocynin. Blockade of ETA receptor (BQ123) and NOX (gp91ds-tat) completely inhibited ET-1-induced ICl,swell in HL-1 cells. These data indicate that ICl,swell is present in HL-1 cell and regulated by similar mechanisms as in native cells. Finally, we confirmed the production of ROS by ET-1 signaling by flow cytometry of HL-1 cells using the nominally H2O2-selective fluorescent probe C H2DCFDA-AM. Exposure to ET-1 increased ROS production, as did H2O2, a positive control. ET-1-induced ROS production was fully suppressed by both gp91ds-tat and rotenone. HL-1 cell ROS production also was stimulated by the mitochondrial complex III inhibitor antimycin A, and antimycin A-induced ROS production was blocked by rotenone but not by gp91ds-tat. These data suggest that ET-1 ETA receptor signaling elicits ICl,swell by sequentially stimulating ROS production by NOX and mitochondria. ETA receptor signaling is down stream from AngII in the osmotic swelling-induced activation of ICl,swell and is upstream from EGFR kinase and PI-3K. Endothelin signaling is likely to be an important means of activating ROS production and ICl,swell in a variety of cardiovascular diseases.

Thesis (Ph. D.)--University of Virginia, 2007. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

L'apéline est l'agoniste du récepteur APJ-R (putative angiotensin II receptor like), récepteur couplé aux protéines G. Exprimée dans le coeur de plusieurs espèces dont l'homme, l'apéline joue un rôle important dans le système cardiovasculaire. L'apéline a un effet inotrope positif sur le coeur. Szokodi et al. ont démontré que cet effet était dû à l'activation de la cascade PLC-PKC et que les échangeurs sodium-hydrogène (NHE) et sodium-calcium (NCX) étaient impliqués. Ils ont également démontré que l'augmentation de la force contractile du myocarde n'était pas due à l'augmentation du courant calcique de type L (I[indice inférieur CaL]). Nous proposons que l'augmentation de la contractilité est due à un effet de l'apéline sur le courant sodique rapide (I[indice inférieur Na]). Matériel et Méthodes. La localisation du récepteur APJ-R fut faite par immunofluorescence sur des myocytes isolés du ventricule gauche de chien. Les mesures du courant sodique sur ces mêmes cellules furent faites par la méthode de patch-clamp en configuration cellule entière en voltage imposé. L'apéline 13 et l'apéline 17 furent perfusées à des concentrations de 100 nM pendant 20 minutes pour évaluer leurs effets sur le courant sodique. Résultats. Le récepteur APJ-R est localisé sur la membrane sarcoplasmique des myocytes au niveau des bandes Z, une structure clé pour la contraction, ce qui nous indique que la localisation du récepteur est propice à la modulation de la contraction cardiaque. L'apéline 13 et l'apéline 17 augmentent I[indice inférieur Max] du courant sodique de 39% et 61% respectivement comparativement au contrôle. En plus de l'augmentation du courant sodique, les deux formes d'apéline déplacent l'activation du canal sodique de -6,8 mV et -8,6 mV pour l'apéline 13 et 17 comparativement à la condition contrôle. Ce déplacement de l'activation vers des potentiels plus négatifs augmente l'excitabilité des myocytes cardiaques et pourrait ainsi moduler la contraction cardiaque. L'inactivation du canal sodique n'est pas modifiée par la présence des deux formes d'apéline. L'apéline ne modifie donc pas la disponibilité du canal en fonction du voltage. Le temps de réactivation est significativement augmenté par la présence d'apéline 13 et 17 ce qui a pour effet d'augmenter la période réfractaire au niveau du coeur. Conclusion. Le récepteur APJ est bien présent au niveau des bandes Z des myocytes suggérant son implication dans la contraction cardiaque. Nos résultats sur le courant sodique démontrent pour la première fois que l'apéline affecte significativement ce courant et que cette augmentation du courant sodique pourrait être responsable de l'augmentation de la contraction cardiaque par l'apéline.

The role of cyclic guanosine monophosphate (cGMP) dependent protein kinase (PKG) in necrotic and apoptotic pathways of many cell types is well established; however its role in the ischemic preconditioning (IPC) of cardiomyocytes is not clearly defined. In the current study, we assessed the hypothesis that PKG protects against cell death following ischemidreperfusion injury in myocytes subjected to IPC. Freshly isolated adult rat ventricular myocytes were subjected to IPC by incubating in ischemic buffer for 30 minutes (min) followed by incubation in normal medium for 30 min. Prolonged simulated ischemia (SI) was created by incubating myocytes in the ischemic buffer for 90 min and reoxygenation (RO) for 120 min in the normal medium. Necrosis was determined by trypan blue exclusion and apoptosis was assessed by TUNEL assay. IPC reduced necrosis as shown by significant decrease in trypan blue positive cells as compared to virgin non-preconditioned myocytes subjected to SI and RO alone (p<.01). Similarly, the number of TUNEL positive myocytes following SI and 18 hrs of RO were significantly reduced in the IPC group. Treatment with PKG inhibitor, KT5832 (2pM) completely abolished the protection against necrosis by IPC. However, KT5832 failed to abolish the protective affect of IPC against apoptosis. Furthermore, myocytes infected with an adenoviral construct of PKG-la (1 x 1 o4 particles/cell) significantly reduced the number of trypan blue and TUNEL positive cells. These results suggest that the PKG signaling pathway plays an essential role in the preconditioning of myocytes against necrosis following SI / RO injury. Furthermore, while the overexpression of PKG protects myocytes against necrosis, as well as apoptosis, IPC may not induce a sufficient level of PKG during 18 hours of RO to induce protection against apoptosis.

L'objectif de ce travail a été de comprendre comment les canaux ioniques sont adressés, organisés et régulés dans des domaines spécialisés de la membrane plasmique des cardiomyocytes. Parmi les partenaires des canaux, les protéines MAGUK (Membrane Associated GUanylate Kinase) sont spécialisées dans l'ancrage, l'agrégation et la formation de complexes macromoléculaires à la membrane. J'ai caractérisé pour la première fois au niveau cardiaque une de ces protéines MAGUK, la protéine CASK. CASK est localisée à la membrane latérale des cardiomyocytes et exclue des disques intercalaires, zones de conduction privilégiée dans l'axe longitudinal. À la membrane latérale, la protéine CASK est exprimée au sein du complexe costamérique dystrophine/glycoprotéines. L'inhibition de CASK entraîne l'augmentation du courant sodique INa dans les HEK293 et les myocytes cardiaques. Dans les HEK293, la microscopie à onde évanescente (TIRF) et des expériences de biotinylation ont mis en évidence que cette augmentation du courant INa est associée à une augmentation du nombre de canaux NaV1.5 à la membrane. La microscopie de conductance ionique (SICM) couplée au patch clamp en configuration cellule attachée a permis de montrer que CASK retient les canaux sodiques au niveau des crêtes et prévient leur agrégation en clusters dans les T-tubules. Enfin, l'inhibition de CASK in vivo par une stratégie reposant sur l'utilisation de virus adéno-associés (AAV) est responsable d'un allongement de la durée de dépolarisation ventriculaire et de l'apparition d'une cardiopathie dilatée. / The aim of the thesis was to understand how ion channels are addressed, organized and regulated in specialized domains of the plasma membrane of cardiac myocytes. Among these partners, the MAGUK proteins (Membrane Associated GUanylate Kinase) are specialized in anchoring, aggregation and clustering of macromolecular complexes at the plasma membrane. In particular, characterized for the first time at the level of the hearth, one of these MAGUK proteins is the CASK protein. CASK is localized at the lateral membrane of cardiomyocytes, but excluded from intercalated disks which are privilege zones of the longitudinal axial conduction. At the lateral membrane, CASK protein is expressed among the costameric dystrophin/glycoproteins complex. CASK inhibition leads to the increase in sodium current density in HEK293 cells and in cardiomyocytes. In HEK293, evanescent wave microscopy (TIRF) and biotinylation experiments pointed out that the INa increase is associated to an increase in the number of NaV1.5 channels at the plasma membrane. Scanning ion conductance microscopy (SICM) coupled to cell-attached patch-clamp has demonstrated that CASK holds together sodium channels at the crest level and prevents their aggregation into clusters in the T-tubules. Finally, inhibition of CASK, in vivo, using an adeno-associated virus strategy resulted to an increase in duration of ventricular depolarization and to the appearance of dilated cardiomyopathy.

The mammalian heart is a remarkable organ in that it must provide for the cardiovascular needs of the organism throughout life, without pausing. Yet, through developmental growth to adulthood and into ageing, the mammalian heart undergoes extensive physiological, morphological and biochemical remodelling. Pivotal to the age-associated alterations in cardiac phenotype is a decline in the proliferative capacity of cardiac myocytes (CMs), which is insufficient to compensate for the basal rate of CM death over time. The terminally differentiated nature of adult CMs also underlies the inability of the heart to repair itself after myocardial damage, such as infarction. As a consequence, existing CMs mount a compensatory hypertrophic response to sustain cardiac output. In parallel, the proliferation rate of resident cardiac fibroblasts, which comprise approximately 60% of total cardiac cells, increases, replacing healthy myocardium with fibrotic scar tissue. Together, CM hypertrophy and fibroblast hyperplasia progressively reduces cardiac function and the ability of the heart to adapt to environmental stressors or damage. Under continued stress or through natural ageing, the heart progresses to a failing state in which cardiac output can no longer meet the demands of the body. The societal impact of ageing-associated decline in cardiac function is great, with heart failure affecting around 8% of over 65s and consuming approximately 2% of the NHS budget. These statistics are set to rise with an ageing population. The substantial phenotypic alterations characteristic of ageing and disease-associated cardiac remodelling requires a wholesale reprogramming of the CM transcriptome. In many biological systems, although yet to be established in adult myocytes, epigenetic mechanisms underlie the transcriptome changes that arise. I hypothesised that alterations in the epigenetic landscape of CMs mediate the transcriptome remodelling that determines the phenotypic transformations that occur in cardiac ageing, hypertrophy and disease. To test this hypothesis, I examined CM-specific changes in DNA cytosine modifications, long non-coding RNA (lncRNA) expression and histone tail lysine methylation marks – epigenetic marks with central roles in transcriptional regulation in many biological systems. I examined how these changes correlate with alterations in the CM transcriptome during disease and ageing. Understanding how alterations in the transcriptome and epigenome contribute to phenotypic changes using whole tissue data is confounded by the heterogeneous nature of the heart, coupled with ageing and disease-associated changes in relative cellular composition. To overcome this, I validated a method to isolate CM nuclei specifically from post-mortem heart tissue. This method also has the advantage that it could be applied to frozen tissue, allowing access to archived material. LncRNAs are functional RNA transcripts longer than 200 bases are emerging as important regulators of gene expression. Common mechanisms of gene expression regulation by lncRNAs include by antisense suppression, as guide/co-factor molecules to direct chromatin modifying components or splicing factors to locations in the genome. Transcriptome profiling in healthy and failing human CMs identified an increase in expression of the lncRNA MALAT-1, which was consistently observed in rodent models of pathology and in ageing. Loss-of-function investigations revealed a potential anti-hypertrophic function for this lncRNA. Specifically, MALAT-1 knock down in vitro in CMs incited spontaneous hypertrophy with features reflecting pathological remodelling in the heart and hypertrophy induced by pro-hypertrophic mediators in vitro. ix In addition, novel uncharacterised transcripts were identified as differentially expressed in cardiovascular disease, including a lncRNA at 4q35.2, which was found significantly downregulated in CMs from human failing hearts. DNA methylation is a stable epigenetic modification and is generally associated with transcriptional repression. It is established by de novo DNA methyltransferases (DNMTs) in early development to determine and maintain differentiated cell states and is ‘copied’ to daughter strands in DNA synthesis by the maintenance DNMT1. Methylcytosine (MeC) can be subject to further processing to hydroxymethylcytosine (hMeC) through a TET protein-mediated oxidation reaction. This serves as a means to actively remove methylation marks as well as hMeC being a novel epigenetic modification in its own right. For the first time, I identified the cardiac myocyte genome as having a high global level of hMeC, comparable with that in neurones. I also discovered an age-associated increase in gene body hMeC that coincided with the loss of proliferative capacity and plasticity of CMs. In parallel, gene body DNA MeC levels decrease in CM ageing. Both these phenomena in gene bodies corresponded with a non-canonical upregulation in expression of genes particularly relevant to cardiac function. This relationship between gene body methylation and transcription rate is strengthened with age in CMs. Recent work in the laboratory had identified the pervasive loss of euchromatic lysine 9 dimethylation on histone 3 (H3K9me2) as a conserved feature of pathological hypertrophy and associated with re-expression of foetal genes. Concurrently, expression and activity of the enzymes responsible for depositing H3K9me2, euchromatic histone lysine methyltransferases 1 and 2 (EHMT1/GLP and EHMT2/G9a) were reduced. Consistently, microRNA-217-induced genetic or pharmacological inactivation of Ehmts was sufficient to promote pathological hypertrophy and foetal gene re-expression, while suppression of this pathway protected from pathological hypertrophy both in vitro and in mice. In summary, I provide new insight into CM-specific epigenetic changes and suggest the epigenome as an important mediator in the loss of plasticity and cardiac health in ageing and disease. Epigenetic mediators and pathways identified as responsible for this remodelling of the CM epigenome suggests opportunities for novel therapy approaches.

Acute myocardial infarction (AMI) rat model was adopted to test the effect of AngioT in vivo. AngioT was directly injected into the ischaemic region of the left ventricle immediately after the ligation of left anterior descending artery. The densities of vessels in AngioT treated hearts were on average 3-4 folds higher compared with non-treated hearts after two and seven days post infarction. Using TUNEL method, approximately 3-fold lower numbers of apoptotic cardiomyocytes were detected in AngioT treated AMI hearts compared with controls. The infarcted volume estimated by Masson's Trichrome staining was significantly decreased in AngioT treated hearts (27.44%+/-7.34% vs. 39.53%+/-5.97%, p&lt;0.05) compared with control hearts 14 days post infarction. Echocardiography demonstrated that left ventricular ejection fraction and fraction shortening in AngioT treated hearts were significantly improved by 10.4% and 22.3% compared to those in the control hearts 2-day post infarction (p&lt;0.05). These improvements were maintained for 2-week post infarction. / Based on the analysis of rat angiogenesis specific superarray, VEGFb, VEGFc and FGF7, were found to be highly expressed in AngioT treated AMI hearts compared to the controls. The expression levels of survival related genes Bcl2 and Akt1 were increased to 3.3 and 2.8 folds respectively in AngioT treated AMI hearts compared with the controls (both p&lt;0.05). Based on the signal transduction pathway finder superarray, Jak-Stat pathway activators, Interleukin 4 receptor and Interferon regulatory factor 1 (IL4R and IRF1), were found to be highly expressed in the AngioT treated AMI heart. / In conclusion, bioactive angiogenic factors (AngioT) were isolated from Geum. japonicum Thunb. Var. Chinense F. Bolle (GJ). Intra-cardiomuscular injection of AngioT had beneficial effects on the acute myocardial infarction. The underlying mechanisms might be related to the activation of Jak-Stat Pathway and over expressions of angiogenic factors and survival associated genes. The therapeutic properties of AngioT appear entirely novel and may provide a new dimension for therapeutic angiogenesis for the treatment of acute ischaemic heart disease. / In the present study, an angiogenic tannins fraction (AngioT) was isolated from Geum. japonicum Thunb. Var. Chinense F. Bolle using bio-assay guided strategy. AngioT increased the proliferation of human umbilical vein endothelial cell (HUVEC) in culture within 24-hour, 48-hour and 72-hour treatment in a dose-dependent pattern. The EC50 of AngioT on HUVEC was less than 25mug/ml. Conditional media from AngioT treated HUVEC stimulated the proliferation of HUVEC significantly greater than conditional media from non-treated HUVEC. Using 2-dimensional electrophoresis and MALDI-TOF, VEGFa was identified in the AngioT treated conditional medium. / Ischaemic heart disease remains the leading cause of morbidity and mortality in most countries. Severe ischaemia of myocardium induces myocardial infarction and results in an irreversible loss of myocardium. Restoration of coronary blood flow by rapid angiogenesis may offer a direct and effective therapeutic way to intractable ischaemic heart diseases. / Key words. ischaemic heart disease; myocardial infarction; therapeutic angiogenesis; Geum japonicum; apoptosis; tannins; VEGF; Jak-Stat pathway / Gu, Xuemei. / "Sep 2007." / Adviser: Peter Tong Chun Yip. / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4615. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 159-177). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.