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BSCR Autumn Meeting 2004

001 RE-EXPRESSION OF A CONSTITUTIVELY-ACTIVE CYCLIN B1:CDC2 COMPLEX IN ADULT RAT CARDIOMYOCYTES IS SUFFICIENT TO REINITIATE CELL DIVISION

K. A. Bicknell*, G. Brooks.School of Pharmacy, The University of Reading, Reading Berskhire RG6 6AJ

The ability of the myocardium to repair itself following a myocardial infarction is severely impaired by the limited capacity of adult ventricular cardiomyocytes to divide. Consequently, the damaged region of the myocardium is replaced by scar tissue and cardiac function is often irreversibly impaired. We previously have shown that adult cardiomyocytes normally do not divide as a consequence of cell cycle arrest in G0/G1 and G2/M. However, during pressure overload-induced hypertrophy, a partial reactivation of the cell cycle machinery occurs that permits adult cardiomyocytes to progress through the G1/S transition prior to their accumulation in G2/M. Interestingly, expression and activity of the G2/M phase cyclin:CDK complex, cyclin B1:CDC2, is not detected in normal adult cardiomyocytes nor in cardiomyocytes undergoing hypertrophy. Thus, we hypothesised that expression and activity of cyclin B1:CDC2 might limit adult cardiomyocyte proliferation. We have used recombinant adenoviruses to re-express cyclin B1 and/or a constitutively active form of CDC2, CDC2AF, in isolated adult rat cardiomyocytes. Re-introduction of cyclin B1 or CDC2AF alone did not result in an increase in cyclin B1:CDC2 kinase activity nor proliferation in adult myocytes compared to control cultures. However, cyclin B1:CDC2 kinase activity was increased 2.9±0.2-fold in cyclin B1 and CDC2AF co-infected cultures compared to controls. Furthermore, re-expression of both cyclin B1 and CDC2AF in adult cardiomyocytes resulted in a significant increase in the number of tropomyosin-positive myocytes (144%±16%), 24 hours after infection, compared to control cultures (100%±8%, n = 5, p<0.05). Interestingly, an increased number of smaller mononuclear tropomyosin-positive myocytes was observed in cyclin B1 and CDC2AF co-infected cultures (17.5%), compared to controls (7.1%). In this study, we have shown that over-expression of the cyclin B1:CDC2 complex reinitiates cell division in adult cardiomyocytes in vitro. Thus, therapeutic strategies targeting the cyclin B1:CDC2 complex might reinitiate cell division in mature cardiomyocytes in vivo and facilitate myocardial regeneration following injury.

002 A GP91PHOX-CONTAINING NADPH OXIDASE CONTRIBUTES TO THE CONTRACTILE DYSFUNCTION ASSOCIATED WITH PRESSURE-OVERLOAD CARDIAC HYPERTROPHY

D. J. Grieve*, J. A. Byrne, J. Layland, A. C. Cave, A. M. Shah.Cardiovascular Division, King’s College London

Reactive oxygen species (ROS) generated by NADPH oxidases may be involved in the pathophysiology of left ventricular hypertrophy (LVH). However, we recently reported that a gp91phox-containing NADPH oxidase is not essential for LVH in response to chronic pressure-overload. Here, we investigated the role of gp91phox in the development of the associated contractile dysfunction. The advent of microconductance technology has significantly advanced the haemodynamic assessment of gene-modified models of cardiovascular disease. In this study, we describe the application of microconductance analysis of cardiac function to the isolated murine ejecting heart. This ex vivo model is complementary to the previously described in vivo preparation, allows assessment without the potentially confounding effects of anaesthetic or neurohumoral influences, and enables careful control of cardiac loading (particularly preload). We first demonstrated that ex vivo pressure-volume relations in the isolated murine heart (n = 8) are sensitive to changes in myocardial contractility induced by aortic constriction. Comprehensive analysis of cardiac pressure-volume relations (1.4 F Millar microconductance catheter) in isolated ejecting hearts demonstrated significant contractile dysfunction in wild-type (WT) bands vs shams in both the steady-state (LV dP/dtmax: 4941±180 vs 5914±211 mmHg/s; stroke work (SW): 12.7±0.8 vs 18.2±1.1 mmHg.μl/mg; P<0.05) and under variable loading conditions (ESPVR: 3.5±0.3 vs 4.8±0.4; EDPVR: 0.080±0.005 vs 0.064±0.005; P<0.05). However, LV contractile dysfunction was preserved in banded gp91phox-/- mice (eg, SW: 17.7±1.5 vs 19.1±1.0 mmHg.μl/mg; ESPVR: 4.7±0.2 vs 4.7±0.3 mmHg; P = NS). Isolated myocyte contractility (% cell shortening) was also significantly decreased in WT bands (4.4±0.3 vs 5.5±0.3%; P<0.05) but not gp91phox-/- bands (4.5±0.3 vs 4.8±0.2%; P = NS).

Conclusions: Although a gp91phox-containing NADPH oxidase is not required for the development of pressure-overload LVH per se, it contributes to the associated contractile dysfunction via changes which are intrinsic to the cardiac myocyte.

003 ENDOTHELIAL TETRAHYDROBIOPTERIN REGULATES ENOS COUPLING/UNCOUPLING INDEPENDENT OF VASCULAR DISEASE: INSIGHTS FROM TARGETED TRANSGENIC MODELS

J. K. Bendall*, N. J. Alp, T. K. Nicoli, K. A. Rockett, S. Kawashima$, M. Yokoyama$, K. M. Channon.Department of Cardiovascular Medicine, University of Oxford, UK; $The First Department of Internal Medicine, Kobe University, Japan

Introduction: Endothelial dysfunction in vascular disease states is associated with eNOS uncoupling so that O2- is produced instead of NO, possibly due to mismatch between eNOS protein and its cofactor tetrahydrobiopterin (BH4). However, the mechanistic relationship between BH4 availability and eNOS activity remains undefined. In particular, whether relative BH4 deficiency alone is sufficient to uncouple eNOS remains unclear.

Methods and Results: We investigated the stoichiometry of BH4-eNOS interactions in vivo by crossing endothelial-targeted eNOS Tg mice (eNOS-Tg) with mice over-expressing endothelial GTP cyclohydrolase (GCH-Tg), the rate limiting enzyme in BH4 synthesis, in which endothelial BH4 levels are increased 3-fold. eNOS protein was increased 8-fold in eNOS-Tg and eNOS/GCH-Tg mice compared with WT. NO synthesis (arginine to citrulline conversion) in lung and aorta, was significantly elevated by 2-fold in eNOS-Tg mice but by 4-fold in eNOS/GCH-Tg mice compared with WT (P<0.05 vs eNOS-Tg). Aortic BH4 levels were depleted 50% in eNOS-Tg compared with WT mice (P<0.05), suggesting oxidative loss of BH4, but BH4 levels were maintained in eNOS/GCH-Tg mice. Endothelial O2- production (oxidative confocal microtopography) was increased 2.5 fold in eNOS-Tg compared with WT aortas but was normalized by L-NAME, suggesting O2- production by uncoupled eNOS. However, in eNOS/GCH-Tg aortas endothelial O2- production was similar to WT, and L-NAME had no effect, indicating preserved eNOS coupling.

Conclusions: These data indicate that eNOS coupling is exquisitely dependent on BH4 levels even in physiological conditions. Thus, strategies to increase eNOS protein without a concomitant increase in endothelial BH4 levels may lead to eNOS uncoupling and paradoxically exacerbate oxidative stress.

004 HYPERTENSION AND ENDOTHELIAL DYSFUNCTION IN MICE FEATURING ENDOTHELIAL CELL-SPECIFIC KNOCKOUT OF ENDOTHELIN B RECEPTORS

A. J. Bagnall*, N. F. Kelland, F. Gulliver-Sloan, G. A. Gray, Y. V. Kotelevtsev, D. J. Webb.Centre for Cardiovascular Science, University of Edinburgh, Scotland. EH8 9XD

Introduction and aims: Endothelin-1 (ET-1) exerts its cardiovascular effects through ETA and ETB receptors (ETBR). Responses to ETBR activation include vasodilatation, vasoconstriction, inhibition of renal sodium reabsorption and clearance of ET-1. Each response may independently modulate blood pressure and blood flow, complicating the interpretation of in vivo pharmacological studies of ETB receptor function. To investigate the contribution of endothelial cell (EC) ETBR to the regulation of blood pressure and vascular tone we have generated mice in which ETBR expression may be spatially regulated.

Methods: Mice featuring loxP sites flanking the ETBR gene (floxed ETBR) were generated by standard gene targeting techniques. Floxed ETBR mice were crossed with Tie2-Cre mice to produce EC-specific ETBR knockout (Flox/Flox Tie2). EC ETBR binding of [125I] ET-1 was measured and wire myography performed to assess endothelial function. Blood pressure (BP) was measured under conscious, unrestrained conditions via in-dwelling catheters in 8–16 week old mice fed with either standard (0.76% NaCl) or high (7.6% NaCl) salt diet. Plasma ET-1 was measured by radioimmunoassay. Wild type (W/W) and single transgenic littermates were used as controls.

Results: EC ET-1 binding was decreased by ∼80% in EC-specific ETBR knockouts (counts/minute/50 μg membrane protein; Flox/Flox Tie2, 581±67; W/W, 3175±268; p<0.001; n = 3). Smooth muscle ETBR-mediated tracheal constriction did not differ between groups. BP was increased in Flox/Flox Tie2 mice (MAP 137.2±6.4 mmHg (n = 5); W/W -/-, 113.7±4.7 mmHg (n = 6; p<0.05) but was unaffected by dietary salt. Plasma ET-1 was increased 4 fold in EC-specific knockouts ([ET-1] pg/ml; Flox/Flox Tie2 12.40±2.95; W/W -/- 2.94±0.83; n = 6;p<0.001). Endothelium-dependent vasodilatation, but not endothelium-independent vasodilatation was impaired following EC ETBR knockout.

Conclusions: Endogenous EC ETBR activation exerts a hypotensive effect that is independent of dietary salt intake and is important for normal endothelial function.

005 THE ROLE OF THE CYCLIN-DEPENDENT KINASE INHIBITOR, P27, IN CARDIAC MYOCYTE HYPERTROPHIC GROWTH

I. C. Kavanagh*, K. A. Bicknell, G. Brooks.School of Pharmacy, The University of Reading, Reading, Berkshire. RG6 6AJ

In order to compensate for increased workload, cardiac myocytes undergo hypertrophic growth to increase their cell size, since they have a limited capacity to divide. At the molecular level, hypertrophic growth involves a partial re-activation of the cell cycle machinery, enabling the cardiac myocyte to advance through the G1/S transition prior to accumulation in G2/M. Cell cycle progression is controlled positively by regulators such as the cyclin-CDK complexes and negatively by regulators such as CDK inhibitors. One such inhibitor is p27 that plays an important role in restricting cell cycle progression at the G1/S border. Previously, we have demonstrated that p27 is down-regulated in response to pressure-overload-induced left ventricular hypertrophy in the rat and, therefore, might play a role in the regulation of hypertrophic growth. To extend our knowledge of how p27 controls hypertrophy, we have treated isolated rat neonatal myocytes with various hypertrophic agonists, including serum and endothelin-1. Hypertrophy was observed with all agonists as confirmed by increases in cell size (microscopy) and the induction of ANF and BNP mRNA expressions. We also demonstrated that the levels of p27 protein were downregulated in neonatal myocytes, in response to these agonists. An ideal model of p27 deficiency is the p27 knockout mouse and we have attempted to use this model to isolate p27-deficient cardiac myocytes in order to study their response to hypertrophic stimuli. However, recent findings from our laboratory and others (Deng et al. 2000. Circulation Research), have demonstrated that wildtype neonatal mouse myocytes undergo autonomous hypertrophy, as determined by increased cell size and total protein levels, when cultured in serum free medium. Therefore, neonatal mouse myocytes are not a good model for studying hypertrophic responses in vitro. Future in vivo studies to compare the effect of pressure-overload-induced hypertrophy between p27+/+ and p27-/- mice, will identify whether myocytes from both backgrounds respond similarly to hypertrophic stimuli.

006 ROLE OF A GP91-CONTAINING NADPH OXIDASE IN CARDIAC MYOCYTE HYPERTROPHY INDUCED BY G-PROTEIN COUPLED RECEPTOR AGONISTS

A. L. Kho*, M. Zhang, A. M. Shah, A. C. Cave.Cardiovascular Division, King’s College London

Although recent studies suggest a critical role for NADPH oxidase-derived reactive oxygen species (ROS) in the pathogenesis of angiotensin-II induced cardiac hypertrophy, the role of the NADPH oxidase in other G-protein coupled receptor (GPCR) agonist-induced hypertrophy is unclear. Thus we have investigated the signalling pathways leading to ROS production and hypertrophy in isolated neonatal rat cardiomyocytes exposed to the GPCR agonists endothelin-1 (ET1, 10 nM) or phenylephrine (PE, 10 μM) or vehicle (serum-free control, SF). Hypertrophy was assessed by cell size and quantitative RT-PCR of atrial natriuretic factor (ANF) mRNA, normalised by β-actin. NADPH oxidase activity was assessed by lucigenin (5 μM) chemiluminescence and in situ ROS production by dihydrochlorofluorescein (DCF) fluorescence.

24 h exposure to PE or ET1 increased cell size and ANF mRNA levels (PE: 13.8±3.4 fold increase vs SF; ET1: 5.3±0.6 fold increase vs SF; P<0.05) concomitant with an increase in NADPH oxidase-dependent ROS production (PE: 90±18%; ET1: 118±21%; both P<0.05 vs SF) and DCF fluorescence (133±41% and 182±49% respectively; P<0.05). The PKC inhibitor, bisindolylomaleimide (Bis, 5 μM), significantly inhibited both PE and ET1-induced NADPH oxidase activation (PE+Bis:17±20%; ET1+Bis: 12±13%; P<0.05 vs PE/ET1 alone), but not PE or ET1 induced increases in ANF mRNA (PE+Bis: 14.3±5.2; ET1+Bis: 13.7±3.1-fold increase, P = NS vs PE/ET1 alone). However, the antioxidant, BHA (butylated hydroxyanisole: 50 μM) significantly reduced PE-induced rises in ANF mRNA (PE+BHA: 3.4±0.6, ET1+BHA: 2.8±1.3; both P<0.05 vs PE/ET1 alone). Transfection with a gp91phox antisense (AS) oligonucleotide had no effect on either PE or ET1-induced DCF fluorescence (PE+AS, 95±22% and ET1+AS 116±20% of the PE/ET1 response alone).

PE and ET1 induce a PKC-dependent activation of NADPH oxidase in myocytes, which does not appear to involve Nox 2. The PE/ET1 induced increase in ANF mRNA is dependent on ROS production, implicating a redox-sensitive signalling pathway. However, failure to inhibit these responses with Bis suggests there are multiple pathways to ANF induction.

007 GLYCATED ALBUMIN STIMULATES OXIDANT STRESS IN CULTURED NEONATAL RAT CARDIAC MYOCYTES: INVOLVEMENT OF GP91PHOX-CONTAINING NADPH OXIDASE

M. Zhang*, A. L. Kho, A. M. Shah, A. C. Cave.Cardiovascular Division, King’s College London, London SE5 9PJ

Background: Nonenzymatic glycation is increased in diabetes. Although several studies have implicated an important role for advanced glycation end products in many of the complications of diabetes, the effects of early-glycation Amadori-modified proteins, such as glycated serum albumin (GSA), remain less well defined. However, in vascular smooth muscle cells, GSA resulted in a significant activation of redox sensitive signaling molecules and a significant stimulation of growth and migration. Thus the aim of this study was to investigate the effects of GSA on reactive oxygen species (ROS) production by cardiomyocytes and to define the underlying enzymatic source(s) and signalling pathways involved.

Methods and Results: Cultured neonatal rat myocytes were incubated with GSA or vehicle (bovine serum albumin, BSA) for up to 24 h. GSA dose-dependently increased in situ ROS production, assessed by dihydrochlorofluorescein fluorescence, with a optimum effect at 400 µg/ml (152±10% versus BSA (100%) at 24 h, P<0.01; n = 3). The earliest significant increase in ROS occurred after 2 h of incubation (128±1%) reaching a maximum at 24 h. Similarly NADPH-dependent O2- generation in cell homogenates was significantly increased at 24 h (179±15% vs BSA (100%); P<0.01, n = 3). ROS production was inhibited by the NADPH oxidase inhibitors apocycin (500 µM) or diphenyleneiodonium (DPI 5 µM), by Tiron (20 mM), but not by L-NAME (100 µM) or rotenone (10 µM). Transfection with gp91phox antisense oligonucleotides significantly reduced GSA-induced in situ ROS production at 24 h (61.4±8.3% decrease, n = 3, P<0.01). The PKC inhibitor, bisindolylmaleimide I (Bis, 5 µM) significantly inhibited AGE-induced NADPH oxidase activation at 24 h (AGE+Bis:120±16 vs AGE:179±15% of control; P<0.05; n = 3). The AGE-induced increase in ROS at 24 h was accompanied by nuclear translocation of NF-κB (inhibited by N-acetylcysteine (NAC)) and an increase in ANF mRNA expression (2.76±0.48 by real-time RTPCR) normalised to BSA control (P<0.05). NAC and apocynin significantly inhibited this increase in ANF mRNA expression (0.96±0.38 and 1.01±0.84 respectively; P<0.05).

Conclusion: GSA stimulates cardiomyocyte ROS production through PKC-dependent activation of a gp91phox-containing NADPH oxidase. The increase in ROS is associated with NF-κB activation and upregulation of ANF mRNA. These findings suggest that early glycated proteins may contribute to cardiac myocyte dysfunction in diabetes.

008 EXOGENOUS TISSUE FACTOR SUPRESSES THE EXPRESSION OF ATRIAL NATRIURETIC FACTOR IN H9c2 RAT CARDIOMYOCYTES

G. A. Frentzou*, C. Ettelaie, A-ML. Seymour.Department of Biological Sciences, University of Hull, Cottingham Rd., Hull, HU6 7RX

The adaptive response of the heart to chronic mechanical overload, termed “cardiac hypertrophy” is an important risk factor in the development of heart failure. During the onset of cardiac hypertrophy, tissue factor (TF), the initiator of the extrinsic pathway of blood coagulation, is shown to be upregulated in the myocardium. We investigated the influence of different concentrations of exogenous human recombinant TF (5 nM, 50 nM, 500 nM and 2 µM) on the expression of atrial natriuretic factor (ANF) in H9c2 rat cardiomyocytes over a period of 15 days, in the presence of 10% foetal calf serum (FCS). The cells were harvested at 1, 2, 5, 10, and 15 days, and analysis of ANF expression carried out by semi-quantitive RT-PCR and western blotting. The ANF expression peaked on the first day (3 and 5 folds for mRNA and protein respectively) and was subsequently suppressed (down to 20% of the control) over the period of investigation. Our data indicate that exogenous TF either alone, or in combination with serum factors, is capable of inhibiting the expression of ANF. This suggests that TF may play a crucial role in cellular homeostasis.

“The support of National Heart Research Funds is acknowledged”

009 UP-REGULATION OF UROKINASE (uPA) MAKES MONOCYTES HIGHLY FIBRINOLYTIC

J. Humphries*, J. Gossage, B. Sawyer, K. Burnand, A. Smith.Academic Department of Surgery, Kings College London, SE1 7EH

Background: The balance between uPA and plasminogen activator inhibitors (PAI) may determine the fibrinolytic balance of the monocyte. The aim of this study was to over-express uPA and determine the effect on monocyte fibrinolytic activity.

Methods: Monocytes were isolated from 8 normal individuals and pre-treated with macrophage colony stimulating factor.These cells were then transfected with 1000 multiplicity of infection (MOI) of uPA expressing adenovirus (ad-uPA) or a blank adenovirus control (ad-0). Cells were harvested 4 days post transfection. Cell conditioned media were analysed for uPA, tissue-type plasminogen activator inhibitor (tPA), urokinase plasminogen activator receptor (uPAR), PAI-1, and PAI-2 using ELISA assays. uPA activity was also estimated by quantifying zones of lysis on a fibrin plate. Total soluble protein and cell viability were assessed using colorimetric assays.

Results: Transfection of monocytes with ad-uPA raised median uPA antigen production by 400 fold compared to zero uPA production in all ad-0 treated controls (p = 0.03). A 160 fold increase in median uPA activity, as determined by fibrin plate lysis, was observed in cells with ad-uPA. No uPA activity was measured in any ad-0 treated cells (p<0.0001). No tPA could be measured in monocytes irrespective of treatment. Up-regulation of uPA decreased PAI-2 production (median 448 (range 140–787) ng/mg soluble protein) compared to controls (median 1000 (range 135–1000) (p = 0.06)). Treatment with ad-uPA also resulted in a significant increase in monocyte uPAR production (p = 0.03) and a significant decrease in PAI-1 production (p = 0.03) when compared to ad-0 treated controls. Adenoviral transfection did not affect cell viability.

Conclusion: Treatment of monocytes with ad-uPA increased monocyte uPA and uPAR production and also decreased production of its main inhibitors. These fibrinolytic cells are being infused into models of deep vein thrombosis to ascertain whether they might accelerate thrombus resolution.

010 ALTERNATIVE SPLICING OF SERUM RESPONSE FACTOR

A. R. Gordon*, J. Metcalfe, P. R. Kemp.Department of Biochemistry, University of Cambridge, Cambridge

Serum response factor (SRF) is a transcription factor that controls smooth muscle specific gene expression and plays a key role in determining the phenotype of smooth muscle cells (SMC). Alteration in the differentiated state of SMC is a common feature of many cardiovascular diseases and changes in SRF expression have recently been implicated in the development of cardiovascular disease. Overexpression of SRF or of a mutant inhibitory form of SRF in mice resulted in increased heart to body weight ratios and bi-ventricular enlargement. Furthermore, recent studies have shown that SRF is alternatively spliced into four isoforms (SRF-L, SRF-M, SRF-S & SRF-I) and that the pattern of SRF splicing is altered in heart failure. The aim of this study is to investigate the effect of SRF splicing on SRF activity and to determine the role of SRF splicing in the control of cell phenotype during normal cardiovascular development and disease.

C2C12 myoblasts and P19 embryonic carcinoma cells were transfected with SRF-L and SRF-M. SRF-M, which lacks part of the transactivation domain, had reduced activity compared to the full length SRF-L. Furthermore, the expression of SRF-M compared to SRF-L in these cells was correlative with the expression of muscle-specific genes. Thus, the relative proportions and activities of SRF-L and SRF-M may have a significant role in the control of muscle-specific gene expression.

To investigate this further, we have generated targeting constructs which will be used to generate mice that are unable to splice SRF into isoforms SRF-L and SRF-M and will therefore lack expression of these proteins. As well as global knockout of SRF-L, we have floxed the gene which will allow for the tissue-specific deletion of SRF-L. Tissue specificity will be achieved by crossing mice with SM-MHC-Cre and MLC2v-Cre mice to generate smooth muscle and cardiac specific knockouts respectively. Analysis of mice will determine if lack of specific SRF isoforms contributes to an abnormal cardiovascular phenotype.

011 THE EFFECT OF TUMOUR NECROSIS FACTOR α INFUSION BY IMPLANTABLE PUMP ON MURINE CARDIAC FUNCTION

M. E. Faircloth*, J. E. Clark, K. Dighe, M. S. Marber.Cardiovascular Division, KCL, Rayne Institute, St Thomas’ Hospital, London SE1 7EH

Introduction: Elevated concentrations of Tumour Necrosis Factor α (TNFα) occur in chronic heart failure, and are associated with poor left ventricular (LV) function and outcome. In mice, cardiac restricted TNF expression leads to chamber dilatation but it is not known whether TNF infusion results in a similar phenotype.

Aims: We have assessed the LV characteristics in mice infused for seven days with TNFα or diluent.

Methods: Osmotic infusion pumps were implanted subcutaneously in 12 male C57B6 mice. Group 1 (n = 6, weight 26.2 g) received recombinant murine TNFα at a rate of 40 or 80 µg/kg/min, and group 2 (n = 6, weight 28.3 g) received 0.9% saline. After seven days, cardiac function in the two groups was assessed by the use of a miniature conductance catheter passed into the LV via the carotid artery, crossing the aortic valve.

Results: No differences were observed between groups for mouse weight, cardiac output (CO), ejection fraction (EF), stroke work, or end-diastolic pressure. There was a slight increase in heart rate (HR) in the TNF group (p<0.05), in which was also observed a marked increase in end-systolic and end-diastolic volumes (p<0.01 for both), and a reduction in end-systolic pressure, dP/dtmax and dP/dtmin (p<0.05 for all). A trend was observed towards a reduction in the maximal elastance and end systolic and diastolic elastance.

Conclusions: This first in vivo mouse study of the effects of prolonged TNF infusion demonstrates LV dilatation. The maximum and minimum rates of change in pressure, measures of contractile function, are reduced. These are characteristic changes of the failing heart, and support the hypothesis that TNF is involved in the causation of heart failure, rather than rising in response to it, and this is supported by the change in the elastance. That the SV, CO and EF have not been seen to be different between groups perhaps reflects early, partially compensated heart failure, a theory further supported by the slight rise in heart rate.

012 P38-MAPK MEDIATES THE EARLY NEGATIVE INOTROPIC EFFECT OF TUMOR NECROSIS FACTOR-α. EVIDENCE OF SYNERGY BETWEEN A DIRECT NEGATIVE INOTROPIC EFFECT AND CORONARY CONSTRICTION

M. Bellahcene*, X. B. Cao, J. Layland, M. Tanno, A. M. Kabir, R. S. Haworth, M. Avkiran, M. S. Marber.Cardiovascular Division, KCL, The Rayne Institute, St Thomas’ Hospital, London SE1 7EH, UK

Background and goal: Tumor necrosis factor-α (TNF) exerts a negative inotropic effect on the myocardium from various species. TNF activates p38-MAPK and this kinase is thought to depress contractility in a calcium-independent manner. We therefore examined TNF effects on contractility in mice lacking the p38-MAPK activator, MKK3.

Methods: The left ventricular developed pressure (LVDP; isovolumic contraction), coronary flow, p38-MAPK and HSP27 phosphorylation, as well as the end-diastolic v LVDP (or Frank-Starling) relationship were analyzed in C57BL/6 outbred, mkk3 wild-type (WT), and mkk3 knock-out (KO) isolated mouse hearts exposed to 10 ng/ml TNF i.c. for 15 min after 40 min of stabilization. Some hearts received SB203580 (p38-MAPK catalytic site inhibitor; 1 μM) for 20 min starting 5 min before TNF infusion. All protocols were run under constant pressure and constant flow conditions since TNF also has vasoconstrictive properties.

Results: TNF (10 ng/ml) for 15 min significantly reduced LVDP and coronary flow in outbred and mkk3-WT mice. This early negative inotropic effect of TNF was associated with a significant phosphorylation of both p38-MAPK and HSP27. However, TNF did not reduce the LVDP and did not phosphorylate p38-MAPK and HSP27 in mkk3-KO mice despite some reduction in coronary flow. Similarly, TNF caused a significant depression of the Frank-Starling relationship in both outbred and mkk3-WT, but not mkk3-KO mice. Furthermore, SB203580 attenuated TNF-induced negative inotropy, as well as p38-MAPK and HSP27 phosphorylations. Comparing constant pressure vs constant flow models of heart perfusion, we found that TNF-induced coronary constriction can act in synergy with a direct effect of TNF on cardiomyocytes to cause p38-MAPK activation, and consequent negative inotropy.

Conclusion: Our results implicate p38-MAPK in the early and dual negative inotropic effect of TNF, and may be of relevance to the pathogenic actions of this cytokine in heart failure.

013 PROTECTION AGAINST ENDOTOXEMIA-INDUCED CONTRACTILE DYSFUNCTION IN MICE WITH CARDIAC-SPECIFIC EXPRESSION OF SLOW SKELETAL TROPONIN I

J. Layland*, A. C. Cave, D. J. Grieve, E. Sparks, J. C. Kentish, R. J. Solaro, A. M. Shah.Cardiovascular Division, King’s College London and University of Illinois at Chicago

The intrinsic impairment of cardiomyocyte contractility characteristic of gram negative endotoxaemia has been partly attributed to a reduction in myofilament Ca2+-responsiveness and has also been associated with increased cardiac troponin I (cTnI) phosphorylation at serines 23 and 24 (residues required for the protein kinase A (PKA)-dependent reduction of myofilament Ca2+-sensitivity following β-adrenoceptor stimulation). To investigate the functional significance of increased TnI phosphorylation in endotoxemia, we studied the contractile effects of systemic bacterial lipopolysaccharide (LPS) treatment in transgenic mice (TG) with cardiac-specific replacement of cTnI by slow skeletal TnI (ssTnI, lacking PKA phosphorylation sites) and matched non-transgenic littermates (NTG) on a CD1 background. In wild-type CD1 mice, LPS treatment (6 mg/kg, IP, 16-18 hours) caused a 37% reduction in isolated myocyte unloaded sarcomere shortening from 6.1±0.2 to 3.9±0.2% (1 Hz, 32°C, P<0.05). Similarly, in NTG myocytes, endotoxemia reduced myocyte shortening by 42%, from 6.7±0.2% to 3.9±0.1% (P<0.05) with no change in intracellular Ca2+ transients. However, in the TG group, LPS reduced myocyte shortening by only 13% from 7.5±0.2% to 6.5±0.2% (P<0.05). LPS treatment significantly reduced contractility during β-adrenergic stimulation (10 nM isoprenaline) in NTG myocytes but not in TG myocytes, even though isoprenaline-induced increases in Ca2+ transient amplitude were similar in both groups. Investigation of the sarcomere shortening-Ca2+ relationship in Triton-skinned cardiomyocytes revealed a significant reduction in myofilament Ca2+-sensitivity following LPS treatment in NTG myocytes, an effect that was substantially attenuated in TG myocytes. In conclusion, the replacement of cTnI with ssTnI in the heart provides significant protection against endotoxemia-induced cardiac contractile dysfunction, most probably by preserving myofilament Ca2+-responsiveness by preventing TnI phosphorylation of PKA-sensitive sites.

014 NO ROLE FOR CALCINEURIN IN SENSITIVITY TO ISCHEMIA/REPERFUSION, MAPK/SAPK ACTIVATION OR ISCHEMIC PRECONDITIONING IN THE EX VIVO MOUSE HEART

K. Obasanjo-Blackshire*, O. Beuno†, J. Molkentin†, M. Marber, R. Heads.Cardiovascular Division, KCL, UK and †Children’s Hospital Medical Center, Cinncinnati, USA

The Ca2+-dependent phosphatase calcineurin A (CnA) plays an important role in inflammation, protection and injury in cardiomyocytes, embryonal heart development and in the development of hypertrophy and heart failure in adults. The developmental and hypertrophic effects of CnA are partly mediated at the transcriptional level via regulation of the NFAT family of transcription factors. CnA may also modulate the activities of several signal transduction pathways including MAPKs and SAPKs. In this study we have used hearts from wild type (WT) mice or mice with targeted disruption (KO) of the CnAβ gene (the major cardiac isoform) to determine differences in sensitivity to ischemia/reperfusion (I/R), in MAPK/SAPK activation and in ischemic preconditioning (IPC) which may be attributable to CnAβ. Intraperitoneal injection of LPS into mice 24 hours prior to removal of the heart and analysis of iNOS expression revealed robust iNOS expression in WT hearts which was abolished in CnAβ KO hearts, suggesting a role for CnAβ in inflammatory signalling. Hearts from outbred C57BL6 mice subjected to 45 min global ischemia (I) and 90 min reperfusion (R) in Langendorff mode showed biphasic transient activation of p38-MAPK, during both I and R, whereas both p42/p44-MAPK and JNK (SAPK1) were activated by reperfusion only. No differences in this pattern of MAPK/SAPK activation were seen in either WT or CnAβ KO hearts. There were also no differences in haemodynamic parameters either at baseline or during I/R, or in infarct size between WT and CnAβ KO hearts. The development of acute IPC in response to 6 cycles of 4 min I/6 min R prior to index I/R was also unaffected (WT 17.3±2.2%* v 31.9±4.3% and KO 14.2±1.5%* v 32±3.2%; *p<0.05 vs I/R by ANOVA). These results suggest that despite an effect on the expression of inflammatory mediators in vivo, CnAβ does not play a role in the sensitivity to ischemia/reperfusion, MAPK/SAPK activation or acute IPC in the buffer perfused heart ex vivo.

015 ARE ELAFIN OVEREXPRESSING MICE PROTECTED FROM ACUTE MYOCARDIAL ISCHAEMIA/REPERFUSION INJURY?

J. Sharif., M. Sallenave, G. A. Gray.Centre for Cardiovascular Science; *Centre for Inflammation Research, College of Medicine and Veterinary Medicine, University of Edinburgh. Edinburgh EH8 9JZ

It has been suggested that early local activation of neutrophil derived serine elastases may play an important role in later myocardial remodelling. Recent studies have demonstrated that overexpression of the serine elastase inhibitor elafin improves myocardial function in models of viral myocarditis (Zaidi et al., 1999) and chronic myocardial infarction (Ohta et al., 2004).

As neutrophils play a major role in cardiac reperfusion injury, the aim of the present study was to investigate whether mice overexpressing elafin (under control of the preproendothelin promoter, EE mice) would be protected from damage in an acute model of myocardial ischaemia-reperfusion.

11-13 week old male C57Bl6 or EE mice (backcrossed onto a C%&Bl6 background) were anaesthetised (ketamine/xylazine, i.p.) and instrumented for measurement of blood pressure. Tracheotomy was performed to permit artificial ventilation. The left coronary artery was ligated for 45 minutes, followed by 2 hours of reperfusion. The ischaemic area (area at risk, AAR) was assessed by infusion of Evans Blue dye at the end of the experiment and ischaemic damage was later assessed in heart sections using tetrazolium chloride.

In control C57Bl6 mice the AAR was 57±9% of the left ventricle and the necrotic zone comprised 38±6% of the AAR. Neither the AAR (63±3%) nor the necrotic zone (40±6%) were significantly modified in EE mice.

The lack of modification of the necrotic zone in the surrent study, together with the observed lack of change in infarct size in chronically infarcted mice (Ohta et al., 2004) suggest that elafin overexpression may target processes other than those involved in immediate ischaemia related damage in the myocardium.

• Ohta K, et al. Am J Physiol 2004;87(1):H286–92.

• Zaidi SHE, et al. J Clin Invest103:1211–9.

This study was supported by the British Heart Foundation PG/03071

016 HYPERTENSION AND CARDIAC HYPERTROPHY IN BTEB3 NULL MICE

M. Keramatipour, C. A. Goddard, W. H. Colledge, P. R. Kemp*.Department of Biochemistry, University of Cambridge, CB2 1QW

We have previously shown that the Krüppel-like factor BTEB3 is expressed in vascular smooth muscle cells and activates the SM22α promoter in vitro by binding to CG rich sequences in the promoter. Using a similar approach we have also found that BTEB3 like similar KLFs activates the cardiac troponin C promoter but can also act as an inhibitor of gene expression including the SM α-actin promoter. BTEB3 is widely expressed in the mouse embryo with strong expression in the heart at E11.0. To determine the role of BTEB3 in the mouse we have generated mice in which exon 1 of the BTEB3 gene is replaced by a neomycin resistance gene.

Genotyping of 246 live born mice has shown that BTEB3-/- mice are born but are under-represented (by a factor of 50%) in the litters at the time of genotyping. The surviving mice are fertile and the litters produced are of a reasonable size. However, the mice show an increase in the ratio of heart weight to body weight (approximately 20%) both at 3 months and at 6 months regardless of whether they are born to -/- parents or +/− parents. This cardiac hypertrophy is accompanied by an increase in systolic blood pressure of approximately 20 mm Hg. Preliminary analysis of the hearts is consistent with dilatation of the right ventricular chamber.

To determine the mechanisms behind these changes the cardiovascular system we are analysing gene expression in the right and left ventricles and in the aorta. The cause of embryonic lethality is also currently under investigation.

017 IDENTIFICATION OF GENES THAT ARE DIFFERENTIALLY EXPRESSED IN HUMAN HEART FAILURE USING QUANTITATIVE REAL TIME POLYMERASE CHAIN REACTION (QRT-PCR)

A. B. Goulter*, J. C. Allen, K. L. Clark.Pharmagene Laboratories, Royston, Herts, SG8 5HD, UK

The use of sensitive gene expression profiling techniques applied to human tissue can reveal novel information regarding well studied targets. Therefore, we investigated mRNA expression of a range of established pharmacological targets in samples of human left ventricular free wall classified as non-diseased, idiopathic dilated cardiomyopathy (IDC) and ischemic cardiomyopathy (ICM) (see table below). In every PCR reaction, the level of the housekeeping gene, GAPDH was also determined (no differences were observed in expression levels between the three groups). Atrial natriuretic factor (ANF) and ACE mRNA were evaluated as positive controls and confirmed to be significantly up-regulated in IDC and ICM relative to the control group. Histamine H2 receptor (H2) and phosphodiesterase (PDE) IV (isoform 2) mRNA levels were down-regulated by approximately 2-fold in both IDC and ICM groups. PDE III (isoform 2) was down-regulated by 3 and 5-fold in IDC and ICM, respectively. This study shows for the first time in the human heart, levels of mRNA for H2, PDE III-2 and PDE IV-2 are down-regulated in both IDC and ICM versus non-diseased myocardium.

Conclusion: This study demonstrates the capability of QRT-PCR to reveal novel data regarding the transcriptional regulation of existing drug targets in human heart failure. Further target validation is now warranted.

018 ABSENCE OF α7-INTEGRIN LEADS TO PROGRESSIVE STRUCTURAL CHANGES IN THE HEART

R. Nadif-Savey*, N. Ismayilova$, L. Neyses, U. Mayer$, M. Emerson.Division of Cardiology and $Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Oxford Road M13 9PT

α7β1 is the major laminin-binding integrin in skeletal, heart and smooth muscle. Integrins are essential for normal structural development and signalling in the heart although the role of the α7 subunit remains undefined. We are therefore investigating the role of α7 integrin in heart integrity and function by studying the cardiac phenotype of α7 deficient mice. Gross histological comparisons between wild type (WT) and knock-out (KO) hearts revealed that the α7 deficient hearts had a clearly abnormal structure defined by an elongated vertical-axis and reduced heart weight (195.9±12.4 mg in WT versus 125±7.9 mg* in KO; *P<0.05) at 12 months of age. Echocardiography showed no significant differences in measured parameters at 3 months of age although at 9 months the data was as shown below:

Left ventricular catheterisation of 6 month old mice showed normal heart function in KO animals basally and following β-adrenergic stimulation. We conclude that deletion of the α7 gene causes a novel, age-dependent cardiac phenotype characterised by changes in chamber dimensions and wall thickness. In younger animals, however, haemodynamic performance is not impaired. Further studies are required to fully characterise the functional implications of α7 integrin deletion and to determine the structural and signalling mechanisms through which α7 modifies heart structure and function.

019 REGIONAL CARDIAC ACTIVITIES OF MATRIX METALLOPROTEINASE-2 & -9 FOLLOWING CHRNIC MI IN MICE: THE INFLUENCE OF GENETIC BACKGROUND & OESTROGEN

I. Sharif, S. C. Riley*, R. Leask*, E. Bruce, D. J. Webb, G. A. Gray.Centre for Cardiovascular Science & *Centre for Reproductive Biology, College of Medicine and Veterinary Medicine, University of Edinburgh

Cardiac rupture is a feature of chronic MI in the mouse, that has been linked to activation of metalloproteinases (MMPs) -2 and –9 in the myocardium. We have observed differences in the incidence of rupture in mice with different genetic backgrounds and also a doubling of the incidence of rupture in ovariectomised (ovx) female mice treated with oestrogen (E).

The aim of the present study was to map regional activation of MMP-2 and -9 following MI in the mouse and to investigate the influence of genetic background and E on their activation pattern. 11–13 week old male and ovx females with or without E supplementation underwent coronary artery ligation for induction of MI. At 4 days post-MI, the activity of MMP-2 and -9 were determined by gelatin zymography in the infarcted, border and non-infarcted regions of left ventricle, septum and right ventricle.

Following MI, the activity of both MMPs was most dramatically increased in the infarct and infarct border zones of all hearts studied compared to control mice. In hearts of mixed background mice the increases, particularly in MMP-9 activity were higher compared to those of C57bl/6J hearts. E had no significant influence on the activity of either MMP in control of MI mice.

Enhanced activation of MMP-2 and -9 enzymes in the infarct and border may account for the higher level of cardiac rupture in C57bl/6J-129/SvJ compared to C57bl/6J mice. However, these MMPs are not directly implicated in the increased incidence of cardiac rupture caused by E.

This work is supported by British Heart Foundation (PG/99192 and FS/03114)

020 A GP91-PHOX-CONTAINING NADPH OXIDASE IS CRITICAL FOR ANGIOTENSIN II-INDUCED INTERSTITIAL CARDIAC FIBROSIS

S. Johar*, A. C. Cave, D. J. Grieve, A. M. Shah.Cardiovascular Division, Kings College London

The development of interstitial fibrosis and cardiac hypertrophy may be independently regulated. A gp91phox-containing NADPH oxidase was previously shown to be necessary for development of cardiac hypertrophy in response to subpressor angiotensin II (AngII) infusion. In this study, we investigated its role in AngII-induced interstitial cardiac fibrosis and the contribution of aldosterone to this process. Mice lacking gp91phox (KO) and wild-type mice (WT) received AngII (1.1 mg/kg/day for 14 days) or vehicle via osmotic minipump. A group of WT AngII-infused mice also received spironolactone, an aldosterone antagonist (SPIRO, 200 mg/kg/day) in chow (n⩾6 all groups). AngII induced a similar rise in systolic blood pressure in WT and KO (WT: 107±4 to 161±8 mmHg, P<0.05; KO: 107±2 to 151±5 mmHg, P<0.05) which was unchanged by SPIRO in WT (167±9 mmHg). Interstitial cardiac fibrosis (Massons Trichrome) increased significantly with AngII in WT (7.2±0.7% to 11.5±1.0%; P<0.05) but not in KO (6.0±0.6% to 5.8±1.0%, P = NS). The AngII-induced increase in fibrosis in WT was inhibited by SPIRO (7.5±1.0%). CCCcsdasasd Consistent with these data, expression of fibronectin and procollagen I mRNA increased in AngII-treated WT by 2.9±0.6 and 3.0±0.7 fold respectively (both P<0.05) but not in KO (1.2±0.1 and 1.4±0.3 fold respectively, both P = NS). SPIRO partially inhibited the AII-induced increase in procollagen I (1.9±0.2 fold) but not fibronectin (2.8±0.3 fold) in WT mice. Myocardial NADPH oxidase activity (5 µM lucigenin chemiluminescence) was increased in AngII-treated WT (2.9±0.4 to 5.1±0.4 integrated light units [ILU]; P<0.05) but not with AngII treatment in KO (3.2±0.5 vs 3.5±0.3 ILU; P = NS). Interestingly, SPIRO also inhibited AngII-induced oxidase activity in WT (3.3±0.1 ILU. These data suggest a critical role for a gp91phox-containing NADPH oxidase in AngII-induced interstitial cardiac fibrosis which is independent of blood pressure or hypertrophy per se. At least part of the effect of AngII may be mediated by aldosterone.

021 OVEREXPRESSION OF IGF BINDING PROTEIN-2 IN MICE PROTECTS AGAINST INSULIN RESISTANCE AND OBESITY

S. B. Wheatcroft, P. A. Crossey, M. Modo, S. Williams, J. Miell, V. Ezatt, A. M. Shah, M. T. Kearney*.Cardiovascular Division, King’s College London, UK

Obesity and insulin-resistance are independent risk factors for the development of cardiovascular disease, but the mechanisms underlying these conditions are not fully understood. Insulin-like growth factor (IGF)-I modulates insulin sensitivity and stimulates adipocyte differentiation. IGF binding protein-2 (IGFBP-2) is an important regulator of IGF bioactivity. To test the hypothesis that IGFBP-2 modulates the development of insulin resistance and obesity, we overexpressed IGFBP-2 in transgenic (TG) mice and assessed metabolic parameters in response to aging and high fat feeding. Female TG mice were compared with littermate wild type (WT) controls (n = 6–8 per group). Data are expressed as mean ± SEM.

At 8 weeks of age, fasting glucose and insulin levels, glucose tolerance tests (GTT) and systolic blood pressure (SBP) were similar in TG and WT animals. At 40 weeks, glucose levels were lower in TG mice than WT in a GTT (10.5±0.5 v 12.8±0.4 mmol/L; P<0.05). TG mice were more insulin sensitive than WT controls in an insulin tolerance test. SBP was also lower in TG mice than WT at 40 weeks (123±2 v 134±2 mmHg; P = 0.001). Susceptibility to nutritional obesity was assessed by feeding a high fat diet for 32 weeks. TG mice gained significantly less weight on the diet than WT controls (9.8±2.3 g v 16.8±2.2 g; P<0.05). Peri-gonadal and mesenteric fat depots were smaller in TG than WT animals (3.7±0.8% v 13.5±1.5%;P =  0.002 and 1.0±0.04% v 2.5±0.5%; P = 0.007). Cross sectional fat area, measured by MRI scanning at the level of the kidneys, was also lower in TG mice than WT controls (40±3% v 54±3% of cross sectional area; P = 0.04). Mean fat cell area was lower in TG mice than WT counterparts (285±33 µm2 v 597±18 µm2; P = 0.003).

In summary, overexpression of IGFBP-2 in mice attenuates age-related changes in glucocompetence and blood pressure, and reduces the susceptibility to nutritional obesity. These data demonstrate a novel and important role for IGFBP-2 in metabolic homeostasis. Funded by the British Heart Foundation

022 MITOCHONDRIAL FATTY ACID TRANSPORT GENES ARE DOWN-REGULATED IN CARDIAC HYPERTROPHY

A. S. Akki*, E. Dyer, S. Richardson, A-ML. Seymour.Department of Biological Sciences, University of Hull, Hull, HU6 7RX

Cardiac hypertrophy is a major risk factor in the development of heart failure. It is characterised by cellular remodelling of the heart. Energy provision is modified in the compensated hypertrophied heart, with an enhanced uptake and utilisation of glucose and a consequent reduction in fatty acid oxidation. The aim of this study was to investigate alterations in gene expression of key enzymes involved in mitochondrial fatty acid transport at an early stage of cardiac hypertrophy using an aortic constriction model. Pressure overload hypertrophy was surgically induced in male Sprague-Dawley rats by abdominal aortic constriction. RNA was extracted from control and hypertrophied hearts 9 weeks post-surgery and gene expression probed using RT-PCR and semi-quantitative analysis. The heart weight to tibial length (Hw/T) ratio was used as an index of hypertrophy. Hw/T ratio was 0.37±0.02 in the control animals. In aortic constriction animals with Hw/T ratios ⩽ 0.38, cardiac expression of Peroxisome proliferators-activated receptor α (PPARα) and Carnitine Palmitoyl Transferase1 (CPT1) were up-regulated relative to Glyceraldehyde phosphate dehydrogenase (GAPDH) and Calsequestrin2 whilst that of Carnitine acylcarnitinetranslocase (CAT) was down-regulated. However, in aortic constriction animals with a greater degree of hypertrophy (Hw/T ratios ⩾0.4), expression of PPARα and CPT1 in hearts were reduced and that of CAT enhanced. These results indicate that PPARα expression modulates expression of CPT1 and CAT in an inverse manner. With increasing hypertrophy, changes in expression of PPARα, CPT1 and CAT occur in parallel with a shift from the beneficial phase of compensated hypertrophy to a maladaptive phase. Thus changes in fatty acid metabolism may contribute to the development of heart failure.

023 EFFECT OF CHRONIC CARNITINE SUPPLEMENTATION ON MYOCARDIAL METABOLISM AND FUNCTION IN EXPERIMENTAL URAEMIA

V. Reddy*, S. Bhandari, A-ML. Seymour.Dept. of Biological Sciences, University of Hull, HU6 7RX

Cardiac complications are the leading cause of mortality in patients with chronic renal failure. Secondary carnitine deficiency frequently occurs in haemodialysis patients and can lead to impaired fatty acid oxidation in the myocardium. In addition, carnitine deficiency is associated with cardiac hypertrophy and heart failure. The aim of this study was to determine the impact of chronic carnitine supplementation on cardiac contractile function and oxidative fluxes in experimental uraemia using 13C NMR spectroscopy. Uraemia was induced in male Sprague-Dawley rats via a two-stage 5/6 nephrectomy. L-Carnitine was administered (5 mM, 0.25 µL/hr) continuously via subcutaneous mini-osmotic pumps for 6 weeks post surgery. Isolated hearts were perfused in the isovolumic mode, with simultaneous monitoring of cardiac function and myocardial oxygen consumption. Carnitine supplementation resulted in a significant rise in serum free carnitine concentration in control (55.4%) and uraemic (77.6%) groups (see table). A significant reduction in heart weight: tibia length ratio and decrease in glucose oxidation was observed following carnitine treatment in control and uraemic groups. In conclusion, chronic carnitine supplementation results in a reduction in left ventricular hypertrophy, associated with a switch in substrate selection from glucose to alternate fuels.

024 INVESTIGATION OF THE CLEARANCE OF ENDOTHELIN –1 FROM PLASMA USING A MURINE ENDOTHELIAL CELL SPECIFIC KNOCKOUT MODEL

N. F. Kelland*1, A. J. Bagnall1, F. H. Gulliver-Sloan1, R. E. Kuc2, A. P. Davenport2, G. A. Gray1, Y. V. Kotelevtsev1, D. J. Webb1.1Centre for Cardiovascular Science, University of Edinburgh, Edinburgh; 2Clinical Pharmacology Unit, University of Cambridge, Addenbrooke’s Hospital, Cambridge

Introduction: Inactivation of endothelin type B receptors (ETBR), though selective pharmacological antagonism or genetic mutation increases the circulating concentration of endothelin-1 (ET-1), suggesting that the ETBR plays an important role in the clearance of this peptide. By crossing floxed ETBR mice (FF/NoCre) with Tie2-Cre mice (WW/Tie2-Cre), we have generated an EC-specific ETBR KO mouse (FF/Tie2-Cre) to investigate the relative contribution of the EC-ETBR to the clearance of ET-1, without the confounding effects of receptor deletion in all other tissues.

Methods: Plasma ET-1 concentrations were measured by RIA and endothelin type A receptor (ETAR) and ETBR density was assessed using quantitative autoradiography. Clearance of ET-1 was measured by injecting a bolus of [125I]-ET-1 (0.37 pmol/mouse; 28 kBq/mouse) i.v. under anaesthesia. Arterial blood was sampled over the subsequent 2 minutes and the radioactivity in each sample was measured (cpm).

Results: Plasma ET-1 concentrations were greater in FF/Tie2-Cre samples (12.4±3.0 pg.ml−1) than in WW/-- controls (3.0±0.8 pg.ml−1) (n = 6; p<0.001). Autoradiography revealed significant downregulation of ETBR in EC-rich tissues such as lung of FF/Tie2 animals (80±21 amol.mm−2) compared to WW/-- controls (8±3 amol.mm−2) (n = 4; p<0.05), and preserved levels of ETAR expression despite higher concentrations of plasma ET-1. The AUC for the cpm-time graph was significantly increased in FF/Tie2-Cre mice [235056±21333 cpm.sec] compared to the WW/-- [86726±10968 cpm.sec] (n = 5; p<0.01). ETBR blockade of WW/-- mice resulted in an AUC unchanged from that of untreated FF/Tie2 animals (n = 4).

Conclusions: These studies indicate for the first time that the EC-ETBR is the primary receptor responsible for the clearance of ET-1.

025 POTENT AORTIC VASOCONSTRICTION MEDIATED BY CELECOXIB AND OTHER PGHS INHIBITORS IN THE ABSENCE OF NITRIC OXIDE

P. B. Anning*, B. Coles, V. B. O’Donnell.Department of Medical Biochemistry and Immunology, Wales College of Medicine, Cardiff University, Cardiff, CF14 4XN

Selective PGHS-2 inhibitors can promote thrombotic cardiovascular events through poorly-understood mechanisms. In this study we investigated the effects of PGHS inhibition on control vascular tone by murine thoracic aortic rings, and whether this is modulated by endogenous nitric oxide (NO). Immunohistochemistry indicated expression of both PGHS-1 and –2 in aortic vessel wall. Constriction and relaxation were determined using an isometric myograph and standard pharmacological techniques. Aspirin (ASP; 10μM) was without effect on endothelium-dependent (ACh) dilatation or phenylephrine (PE)-constriction, whilst celecoxib (CXB; 10 μM) had no effect on constriction, but caused a small inhibition of dilation at low dose ACh, with a small enhancement in dilation at the higher ACh concentrations. Finally indomethacin (IND; 10μM) caused a small inhibition of constriction, but had no effect on ACh-dependent dilation. These effects were minor however, and for the most part not significant. In total contrast to their small effects on tone when added alone, co-administration of IND, ASP and CXB with L-NAME caused a large significant enhancement in vasoconstriction, and further enhanced the inhibitory effects of L-NAME on ACh-dependent dilation. Finally, in the presence of PGHS inhibitors, there was a significant shift of the SNP-dependent dilation curve to the left indicating enhanced sensitivity of the contractile apparatus to exogenous NO. These data indicate that in the presence of NO, PGHS isoforms play minor roles in regulation of vascular tone in aorta. However, in the presence of L-NAME, PGHS (probably PGHS-2) promotes vascular relaxation, perhaps to compensate for the absence of NO. Importantly, inhibition of endogenous NO generation converts PGHS inhibitors agents into potent vasoconstrictors. These undesirable effects of PGHS inhibitors may contribute to the vascular side-effects of these drugs observed in patients with vascular disease, who characteristically show decreased NO bioactivity in vivo.

026 CHARACTERISATION OF VASCULAR FUNCTION IN MICE DEFICIENT IN THE COMPLEMENT REGULATOR, CD59A

V. B. O’Donnell*, B. Coles, B. P. Morgan, P. B. Anning.Department of Medical Biochemistry and Immunology, Wales College of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN

There is emerging interest in the role of inflammation in hypertension and angiotensin II-dependent signaling. The terminal complex of complement is an important contributor to the innate immune system which is found in human atherosclerotic lesions, suggesting it participates in the inflammatory response associated with vascular disease. To evaluate the potential role of complement lysis in regulation of vascular tone and angiotensin II-dependent hypertension, vascular function was characterized in mice deficient in CD59a, an important inhibitor of membrane attack by complement, that display constitutively elevated complement lysis intravascularly with a mild haemolysis (Holt et al, Blood, 2001;98:442). In vitro, aortic rings from CD59a-/- mice showed impaired vasoconstriction to phenylephrine, and significantly decreased expression of PECAM-1. However, acetylcholine- and sodium nitroprusside-dependent dilatation was identical to wild-type, and plasma nitrate/nitrite and aortic cGMP levels were unchanged. CD59-/- mice had similar basal blood pressure to wild type, and in vivo infusion with angiotensin II by osmotic minipump (1.1 mg/kg/day) caused a similar degree of hypertension in both strains over 7 days. Furthermore, there were no significant differences in angiotensin II-dependent cardiac or vascular hypertrophy between the strains. These data indicate that elevated vascular complement activity is associated with some alterations in reactivity of large vessels to vasoconstrictors and decreased PECAM-1 expression, however in vivo vascular function, including blood pressure and hypertrophic responses to a pressor dose of angiotensin II appear normal.

027 MICE WITH A KNOCKOUT FOR INDUCIBLE NITRIC OXIDE SYNTHASE ON AN OBESOGENIC DIET ARE PROTECTED FROM METABOLIC BUT NOT VASCULAR DYSFUNCTION

B. T. Noronha, S. B. Wheatcroft, J. M. Li, A. M. Shah, M. T. Kearney.Cardiovascular Division, Kings College London

Obesity an independent risk factor for the development of atherosclerosis is associated with abnormalities of insulin action and endothelial function. This study sought to explore the mechanisms underlying endothelial dysfunction in mice rendered obese using a high fat diet.

Male C57BL/6 mice and iNOS knockout mice on a C57BL/6 background were fed an obesogenic diet (35% fat, 35% carbohydrate) from weaning until 8 weeks. Body weight, epididymal fat pad mass, glucocompetence, systolic blood pressure and vasomotor responses in aortic rings ex-vivo were assessed after 4 and 8 weeks of feeding. Glucose regulation was evaluated by a Glucose Tolerance Test (1 mg/kg i.p) and an Insulin Tolerance Test (0.75 units/ kg i.p) after an overnight fast. Systolic Blood Pressure was measured by tail cuff plethysmography. Vascular responses were studied by suspending thoracic aortic rings (4 per animal) in a temperature controlled organ bath. Dose response curves were plotted for vasoconstriction to Phenylephrine before and 30 minutes after exposure to L-NMMA (a non-specific NOS inhibitor). Similarly dose response curves to acetylcholine (Ach 1 nM-10 μM) were performed before and after exposure to catalase (1250 units/ml), an enzyme that dismutes H2O2.

Results are shown in the table overleaf. Both wild type C57bl/6 and iNOS knockout mice developed comparable degrees of obesity and hypertension. Glucocompetence was however significantly better in the iNOS knockout mice than wild type mice suggesting than iNOS may play a key role in impaired insulin signalling during obesity. Vascular response to insulin (0.1 units/ml) an effect mediated by NO release was lost in wild type mice at 8 weeks but preserved in iNOS knockout mice. Basal NO production in mouse aortic rings was significantly lower in iNOS knockout mice than wild type mice fed high fat diet. Endothelial dysfunction was unmasked by catalase in both groups.

These results suggest that iNOS may play a key role in impaired insulin actions in both skeletal muscle and the vascular wall in obesity, despite being protected against impaired insulin action, mice with a knockout for iNOS had blunted acetylcholine responses compensated for by production of H2O2 in diet induced obesity.

028 TRANSCRIPTIONAL REGULATION OF THE NOX4 ISOFORM SUBUNIT OF NADPH OXIDASE IN ENDOTHELIAL CELLS

*MPitsiouni, A. Brewer, A. M. Shah, C. D. Gove.Cardiovascular Division, King’s College London, UK

A striking correlation exists between increased NADPH oxidase activity, a major source of reactive oxygen species (ROS) in cardiovascular cells, and heart disease. Several Nox isoforms, the catalytic subunit of the NADPH oxidase multi protein complex, have been identified in cardiac cells with distinct patterns of expression. Upregulation of transcription of Nox subunits is suggested to increase NADPH oxidase activity. We have cloned a human genomic fragment comprising putative Nox4 promoter upstream of a luciferase reporter and tested activity in transfected cell lines. Serial deletions of the 2 kb upstream of the human Nox4 gene revealed −300 bps to be the basal promoter in endothelial cells, HMEC-1. Similar promoter activity of the constructs in other cell types suggests that cell specific regulation of Nox4 expression is mediated by elements located elsewhere. Three areas highly homologous between human and mouse were identified within 27 kb upstream of the Nox4 gene indicating possible regulatory significance. These were cloned next to the basal human Nox4 promoter in sense (S) and antisense (AS) orientations. A 1.9 kb (−21/−19 kb) fragment reduced the basal promoter activity by 39.9±0.08% S and 51.9±0.07% AS suggesting the presence of a repressor (n⩾3, ANOVA, Bonferroni, p⩽0.0008). This is the first report of the Nox4 isoform promoter regulation further studies may inform signalling pathways for the regulation of ROS production and therapeutic manipulation.

029 A COMPARISON OF THE HYPOTENSIVE EFFECTS OF CALCITONIN-GENE RELATED PEPTIDE AND ADRENOMEDULLIN IN ANEASTHETISED MICE

N. C. Clark*, S. D. Brain.Dept. of Cardiovascular Biology & Medicine, King’s College London, SE1 1UL

CGRP and the related peptide adrenomedullin (AM) have depressor effects on blood pressure (BP), thought to be mediated through receptors composed of G-protein-linked calcitonin receptor-like (CL) receptors with receptor activity-modifying proteins 1, 2, or 3 (RAMP1, 2 or 3; McLatchie et al. 1998). Although the hypotensive effects of exogenous CGRP and AM are well characterised in many species, there have been few studies in the mouse. Here, the hypotensive effects of CGRP and AM were assessed in naturally ventilated, urethane (2.5 mg/g, i.p.) anaesthetised CD1 and RAMP2 transgenic mice. Agents were given as a bolus injection into the jugular vein, and BP was monitored via a cannula inserted into the carotid artery.

CGRP and AM produced dose-dependent hypotension but CGRP was ∼100-fold more potent than AM. In addition, whilst the responses to CGRP were reproducible with only a 15 min dose interval, those to AM required a >2 h recovery period, indicative of tachyphylaxis. To determine the receptors involved in CGRP- and AM-induced hypotension, mice were pretreated with antagonists at CL/RAMP1 and 2 receptors, BIBN4096BS and/or adrenomedullin (AM)22–52, respectively. While CGRP responses were abolished by BIBN4096BS, those evoked by AM were insensitive to both BIBN4096BS and AM22–52. In unique RAMP2 over-expressing mice CGRP-evoked hypotension was unaltered, whilst the hypotension produced by AM was significantly enhanced in RAMP2 OE mice compared with that produced in WT mice. These data demonstrate the dose-related ability of exogenous CGRP and AM to act as potent but distinct hypotensive agents in the mouse. These results suggest that CL/RAMP1 complexes are critical for CGRP-, but not AM-mediated effects, and that RAMP2 participates in AM- but not CGRP-induced hypotension.

• McLatchie et al. Nature 1998;393:333–339.

N.C. is funded by the B.H.F

030 EVIDENCE FOR THE FUNCTIONAL IMPORTANCE OF RAMP2 IN INFLUENCING ADRENOMEDULLIN VASOACTIVE RESPONSES

C. W. Tam*, Z. Lazar, W. Born, S. D. Brain.Centre for Cardiovascular Biology & Medicine, New Hunts House, Kings College London, Guy’s Campus, London SE1 1UL

The potent vasodilators adrenomedullin (AM), produced by vascular cells, and CGRP, which is structurally related and a neuropeptide, act via the CGRP receptor family. The receptors are formed from the seven transmembrane domain calcitonin receptor like receptor (CL), and one of three single transmembrane domain proteins termed receptor activity modifying proteins (RAMP). Thus, an AM receptor is formed from CL and RAMP2 whilst CL and RAMP1 form a CGRP receptor (McLatchie et al., 1998). This theory has only so far been established in cultured cell lines. Our aim is to investigate this theory in tissues isolated from RAMP2 over expressing (TG) mice. Previous studies in mouse isolated aorta have demonstrated a response to AM that was insensitive to the CGRP peptide antagonist, CGRP8–37, thus suggesting that AM acts via the CL/RAMP2 receptor in this tissue. The AM transgenic mice were generated by insertion of a SigMyc-mRAMP2 cDNA construct into fertilized oocytes and then introduced in to the genome of C57Bl6 mice. Mice negative for the transgene were used as wild type (WT) controls. Sections (∼3 mm) of thoracic aorta were isolated and mounted in a glass organ chamber and tone (>0.2 g) was induced by noradrenaline (100–300 nM). Relaxation was induced in a dose dependent manner in response to CGRP (0.3–30 nM) and AM (0.3–100 nM). The responses to CGRP were similar in both WT and RAMP2 TG mice. By comparison, AM responses were significantly (p<0.05) enhanced in terms of sensitivity and maximal responses. Additional studies using CGRP and AM antagonists provided further evidence for the CL/RAMP receptor theory. Taken together, these results support the theory that RAMP2 has functional relevance in mediating AM responses in vascular tissues.

• McLatchie LM, Lee MG, Foord S. Nature 1998;393:333–339.

C.Tam is funded by the British Heart Foundation.

031 C-REACTIVE PROTEIN IS AN INDEPENDENT PREDICTOR OF ENDOTHELIAL DYSFUNCTION IN CORONARY ARTERY DISEASE PATIENTS OPTIMALLY TREATED WITH STATINS: STUDIES IN HUMAN SAPHENOUS VEIN

A. Momin, A. Shah, D. Grieve, S. Wheatcroft, C. Driver, A. El-Gamel, J. Desai, L. John, M. Marrinan, R. Sherwood, M. Kearney.Cardiovascular Division, King’s College London

Endothelial dysfunction (ED), a key event in the initiation/progression of atherosclerosis, is common in patients with coronary artery disease (CAD) despite control of conventional risk factors. Recently, novel non-lipid risk factors for CAD have emerged, among which C-reactive protein (CRP) has been suggested to be linked to ED. However, the relationship between CRP and ED in CAD patients treated with statins who have optimal plasma cholesterol and blood pressure control is unclear. The aim of this study was to explore the role of CRP as a predictor of ED in patients optimally treated with statins. We recruited consecutive patients with CAD undergoing elective CABG surgery, excluding patients with inflammatory diseases that may lead to elevated CRP. Endothelial function was assessed ex vivo in saphenous vein (SV) rings obtained at time of CABG. Of 115 patients, 99 (mean age 66±0.9; 90% male) were optimally treated with statins, with cholesterol levels <5.0 mmol/L (mean 3.1±0.7) and triglycerides 1.2±0.1 mmol/L. These patients had BMI 26.9±0.4 kg/m2, waist circumference 99.8±1.0 cm, systolic blood pressure 138.7±2.2 mmHg, diastolic blood pressure (DBP) 74.3±1.1 mmHg, fasting glucose 6.2±0.2 mmol/L, and CRP 2.4±0.2 mg/L. Vascular reactivity of preconstricted SV rings was assessed in 77 of these patients. Maximal relaxation to the endothelium-dependent vasodilator acetylcholine (Ach; 10−9–10−4 mol/L) was 25.5±2.3% and to the endothelium-independent vasodilator sodium nitroprusside (SNP; 10−9–10−4 mol/L) was 125.8±7.0%. In univariate analysis, CRP (r = −0.3), waist circumference (r = −0.19) and BMI (r = −0.2) were significantly inversely correlated with relaxation to Ach (all P<0.05). In stepwise multivariate analysis, the only independent predictor of relaxation to Ach was CRP (r = −0.3; P = 0.025). Relaxation to SNP was not associated with any of the variables. In multivariate analysis to assess correlates of plasma CRP in this group of patients, the only independent predictors were BMI (r = 0.34), DBP (r = 0.25) and fasting glucose (r = 0.3) - all P<0.01.

Conclusion: In statin-treated CAD patients with low cholesterol levels undergoing CABG, CRP is the only independent predictor of ED. These data support a link between CRP and ED even in patients treated with current;y recommended doses of statins.

032 LEPTIN IS AN ENDOTHELIAL INDEPENDENT VASODILATOR IN HUMANS WITH CORONARY ARTERY DISEASE: EVIDENCE FOR DIVERGENCE OF RESISTANCE TO LEPTIN’S VASCULAR AND METABOLIC ACTIONS

A. Momin, A. Shah, D. Grieve, C. Driver, R. Sherwood.M Kearney Cardiovascular Division, King’s College London

Leptin has emerged as an important peptide in metabolic regulation, with obese patients often being resistant to the effects of leptin. Recently, studies have suggested that leptin may also have vasoactive effects. We explored this possibility in human patients undergoing coronary artery bypass surgery. The vasoactive effect of leptin was assessed ex-vivo in saphenous vein (SV) rings obtained at time of CABG. 131 patients undergoing CABG were recruited (age 66±0.9, (mean±SEM) 90% male).These patients had body mass index 26.8±0.4 kg/m2, waist circumference 99.2±1.0 cm, systolic blood pressure 138.6±2.0 mmHg, diastolic blood pressure 74.4±1.2 mmHg, fasting glucose 6.5±0.2 mmol/L, LDL cholesterol 1.8±.06 mmol/L, HDL cholesterol 1.0±0.2 mmol/L.Vascular reactivity to leptin (10−13–10−7M) was assessed in preconstricted SV rings in all patients (n = 131). Mean maximal relaxation to leptin was 24.5±1.6%; all rings relaxed fully to sodium nitroprusside. To explore the mechanism of leptin’s vasorelaxant effect we performed studies (n = 8) in the presence of the non-selective nitric oxide synthase inhibitor L-NMMA (10−4M). This had no effect on leptin-induced vasorelaxation (17.4±3.4 versus 17.8±3.3%; P = 0.9). Endothelial denudation also had no effect on leptin-induced vasorelaxation (17.4±4.4 versus 22.5±3%; P = 0.4). Since the central effect of leptin is thought to be mediated through potassium channels, we explored the possibility that its vascular actions are mediated in a similar fashion. In the presence of KCl 30 mmol/L to inhibit hyperpolarisation, the vasodilator effect of leptin was completely blocked (12.6±5.6 versus 0.08±4.1%; P<0.001). To assess the relationship between total body fat and leptin’s vascular effect, we assessed the correlation between body mass index (range 16.5–39.1 Kg/m2) and maximal leptin-induced vasodilatation (range 0–102.9%) in the whole population. However, there was no correlation between these variables (r = 0.05, P = 0.55). These data demonstrate for the first time that leptin is a vasodilator peptide in human veins. Moreover, the actions of leptin are endothelial- and NO-independent. These data also show that the vasoactive effects of leptin are preserved in obese patients, supporting tissue specificity of leptin resistance in humans.

033 PLAQUE RUPTURE-RELATED GENE EXPRESSION IN FAT-FED APOLIPOPROTEIN E KNOCKOUT MICE

N. Al-Shanti*, H. J. McKinnon1, C. J. Long1, D. Dunbar1, D. Mallinson1, C. L. Jackson.Bristol Heart Institute, University of Bristol, Bristol BS2 8HW; 1Organon Laboratories Ltd, Newhouse ML1 5SH

Unstable plaques are ultimately responsible for most myocardial infarctions and strokes. The molecular events that govern plaque stability are poorly defined. A better understanding of these events would aid in the identification of novel therapeutic strategies for cardiovascular disease. To this end, we utilised an apolipoprotein E knockout mouse model which is highly susceptible to spontaneous plaque rupture in the brachiocephalic artery after 8 weeks of high-fat feeding. The left common carotid artery was used as a comparator tissue, as it is resistant to plaque rupture in these mice. In order to compare gene expression in these two arteries, we used Affymetrix microarray technology to scan 12,000 genes across a range of time points (0, 5, 7, 8 and 10 weeks of high-fat feeding). This revealed 504 genes that were significantly differentially expressed. Three of these (vitamin D receptor (VDR), klotho, and CC-10) were selected for further investigation based on their expression patterns, which showed a substantial elevation at 8 weeks in the brachiocephalic artery when compared either with time zero or with the left carotid artery at 8 weeks. These data were confirmed by TaqMan PCR. Immunocytochemical staining, using antibodies validated by Western blotting, showed specific regional distributions of these proteins in unstable plaques. These data indicate that rupturing plaques show specific patterns of gene expression, and suggest novel targets for the development of plaque-stabilising therapies.

034 THE DIFFERING EFFECTS OF MATRIX METALLOPROTEINASES ON ATHEROSCLEROTIC PLAQUE INSTABILITY

J. L. Johnson*, S. J. George, A. C. Newby, G. D. Angelini, C. L. Jackson.Bristol Heart Institute, University of Bristol, Bristol, England, BS2 8HW

Matrix metalloproteinases (MMPs) are thought to be involved in the destabilization and rupture of atherosclerotic lesions. We are testing this hypothesis in apolipoprotein E (apoE) knockout mice that have been crossed with various MMP knockouts and fed a high-fat diet for 8 weeks. The present study focuses on apoE/MMP-3 (stromelysin-1), apoe/MMP-7 (matrilysin-1), apoE/MMP-9 (gelatinase B) or apoE/MMP-12 (macrophage metalloelastase) double knockouts. Plaques in the proximal 150 μm of the brachiocephalic artery were assessed for cross sectional lesion area and the frequency of healed plaque ruptures. In addition the plaque content of smooth muscle cells and macrophages were also evaluated.

These data indicate that MMP-3 and MMP-9 play a protective role, limiting plaque growth and reducing plaque rupture. Conversely, the findings suggest that MMP-12 plays a destructive role, inducing plaque growth and triggering destabilisation. Whilst MMP-7 may promote plaque instability by reducing SMC content. This challenges the concept that MMPs simply degrade matrix and thus destabilise plaques, and suggests that members of the MMP family have diverse effects on plaque stability. Therefore, development of selective MMP inhibitors is essential to enable any potential therapeutic intervention of human atherosclerotic plaque rupture and its fatal sequelae. In particular MMP-12 appears to be an attractive target.

035 DIVERGENT EFFECTS OF ASPIRIN AND CLOPIDOGREL ON PLAQUE PROGRESSION IN THE BRACHIOCEPHALIC ARTERIES OF APOLIPOPROTEIN E KNOCKOUT MICE

H. Williams*, N. Baker§, A. Poole§, C. L. Jackson.Bristol Heart Institute and §Department of Pharmacology. University of Bristol, Bristol BS2 8HW, UK

Aspirin has been suggested to reduce acute cardiovascular events, therefore we have tested it in our apolipoprotein E knockout (apoE KO) mouse model of plaque destabilisation. In order to assess the contribution of aspirin’s platelet inhibitory action to any effect on the plaque, we also tested clopidogrel. ApoE KO mice were fed high-fat diet for 8 weeks, and simultaneously received either aspirin (15 mg/kg/day, n = 65), clopidogrel (1 mg/kg/day, n = 63), or were untreated controls (n = 92). Both aspirin and clopidogrel profoundly inhibited platelet function in these mice (data not shown). Brachiocephalic artery sections were analysed by computerised morphometry. Data are shown in the table below as mean±standard error, and statistical analysis was performed by non-parametric analysis of variance.

Treatment with clopidogrel, but not aspirin, reduced plaque size, while neither treatment had any effect on plaque stability. These data suggest that inhibition of platelet function may reduce plaque progression by reduction of thrombus incorporation following plaque rupture. However as aspirin and clopidogrel inhibit platelet function similarly, we propose that aspirin may have a dual role, by also promoting plaque progression via the 5-lipoxygenase pathway, and that this blunts its beneficial actions.

036 INFLUENCE OF SCANNING FREQUENCY AND ULTRASONIC CONTRAST AGENT ON REPRODUCIBILITY OF LEFT VENTRICULAR MEASUREMENTS IN THE MOUSE

I. Sharif, T. Anderson*, D. Anderson*, D. J. Webb, G. A. Gray, W. N. McDicken*, M. A. Denvir.Centre for Cardiovascular Science and Department of Medical Physics*, University of Edinburgh, Scotland, United Kingdom

Background: The small size and fast heart rates of mice represent technical challenges to echocardiography. This study examined the influence of different scanning frequencies and ultrasonic contrast agent and on the accuracy and reproducibility of measurements of left ventricular structure and function.

Methods: Normal mouse hearts (C57BL6) were imaged under general anaesthesia at three different scanning frequencies before and after intravenous injection of the ultrasonic contrast agent (UCA), Optison®. Coronary ligation mice (CAL) and sham-operated controls (SH) were scanned at 10–22 MHz with and without ultrasonic contrast agent.

Results: Scanning frequency had no significant effect on intra- or inter-observer variation of LV measurements in normal mice under baseline conditions. Use of UCA significantly reduced estimated EF at 10–22 MHz compared to baseline (baseline 50.8±7.6% vs UCA 39.7±7.6%, p = 0.03) and significantly increased values for LV cavity dimensions (e.g. LVarea-diastole, 20.74±1.20 vs 23.23±0.98 mm2, p = 0.002). UCA significantly reduced intra- and inter-observer variation in LV ejection fraction.

Conclusions: Scanning frequency had no significant effect on reproducibility of LV measurements in the mouse but UCA significantly reduced inter-observer variation. Use of UCA could reduce the number of mice required to observe a statistically significant change in left ventricular function due to an experimental intervention.

037 PRESSURE VOLUME RELATIONS IN THE MOUSE USING CONTRAST ENHANCED ECHOCARDIOGRAPHY

I. Sharif, T. Anderson*, D. Anderson*, D. J. Webb, G. A. Gray, W. N. McDicken*, M. A. Denvir.Centre for Cardiovascular Science and Department of Medical Physics*, University of Edinburgh, UK

Background: The end systolic pressure volume relationship (Es) is acknowledged as defining systolic performance of the left ventricle independent of loading conditions. Echocardiography combined with high fidelity LV pressure recordings can be used to determine this relation. However, the reproducibility of this technique in the mouse has not previously been assessed.

Methods: Coronary ligation (CAL, n = 12) and sham operated (SH, n = 9) mice (C57BL6) were imaged under general anaesthesia at 10–22 MHz before and after intravenous injection of the ultrasonic contrast agent (UCA), Optison® during simultaneous measurement of LV pressure using a pressure-transducer tipped catheter (1.4 F, Millar Instrument Inc, USA) inserted via the right carotid artery. Intravenous sodium nitroprusside (10–40 mcg/kg/min) was used to alter loading conditions.

Results: Under baseline conditions CAL mice demonstrated a reduced Es slope (see graph) compared with SH consistent with reduced systolic function. In the presence of UCA the Es slope reduced further in CAL animals but was unchanged for SH. The inter-observer variation in Es slope was imrpoved by the use of UCA.


Graphic
Figure 1

Conclusions: Echocardiography combined with high fidelity pressure recordings in the mouse permits the assessment of the pressure volume relation in the mouse, reproducibility of this measurement was significantly improved by the use of UCA.

038 CONDUCTANCE CATHETER FOR IN-VIVO CARDIAC FUNCTION IN MICE - IS IT SENSITIVE ENOUGH?

K. J. Dighe*, G. S. Kanaganayagam, M. S. Marber.Cardiovascular Division, King’s College London, SE1 7EH

We questioned whether the conductance catheter is a sensitive method to determine in-vivo cardiac function in mice. We conducted pressure volume analysis (Millar catheter, SPR-839) on mice with varying degrees of cardiac dysfunction at 4 or 10 weeks of remodelling (R) following permanent LAD occlusion (∼40%MI) at steady state and at varying preload. See table; Shams  = S.

Conclusion: Although we obtained significant remodelling using parameters recorded at steady state, those recorded under varying preload (such as IVC occlusion until ESP dropped to ∼60 mmHg) seemed less sensitive and specific. It is unclear at present whether this is a result of technique or data acquisition and processing.

039 IS THERE OBSTRUCTION OF THE AORTIC VALVE USING A CONDUCTANCE CATHETER IN A MOUSE MODEL?

K. J. Dighe, M. E. Faircloth, J. E. Clark*, G. S. Kanaganayagam, M. S. Marber.Cardiovascular Division, Kings College London, Rayne Institute, St. Thomas’ Hospital, London. SE1 7EH

Pressure volume loops generated using an ultra-miniature conductance catheter are increasingly used to measure cardiac contractility in murine models. Catheters are introduced in the left ventricle (LV) either by LV puncture or following carotid (ICA) cannulation by crossing the aortic valve (AV). We questioned whether the catheter (Millar Instruments, Texas, USA), although small might cause obstruction when placed across the murine AV since the catheter occupies approximately 45% of the cross-sectional area of the aortic valve. Using the ICA route we simultaneously measured aortic pressure (AoP) using left ICA cannulation and LV pressure (LVP) using either direct LV puncture (DLVP, n = 6) or using right ICA cannulation (ICAC, n = 6). The results are described in the figure.


Graphic
Figure 2

No statistically significant differences were seen between the groups. Furthermore, the trend towards an increased AV gradient in the crossed group is of no functional significance. After this preliminary study with relatively small sample sizes we conclude that this technique does not compromise cardiac function or physiology despite the catheter occupying a significant part of the aortic orifice. This finding ratifies the use of these catheters in murine cardiovascular models. Since these models form an important and increasingly utilised tool in the study of cardiac disease, more credence may be given to results obtained by this technique.

040 SUSCEPTIBILITY OF MITOCHONDRIA TO PALMITATE IS INCREASED IN THE AGED HEART

J. Sample*, J. G. F. Cleland, A-ML. Seymour.Depts of Biological Science and Cardiology, University of Hull, HU6 7RX

Age is a major risk factor for heart failure. However, the underlying cellular mechanisms responsible for this increased susceptibility are yet to be defined. Mitochondria play a central role in cardiac metabolism and apoptotic cell death and dysregulation of both of these processes has been shown to be implicated in the transition to failure. Mitochondrial dysfunction in the senescent heart may contribute to impaired cardiac function and an increased susceptibility to injury. The aim of this study was to investigate the impact of palmitate on the mitochondrial membrane potential (Δψm) in isolated cardiomyocytes. Ventricular myocytes were isolated by collagenase digestion from 6 (n = 4) and 24 (n = 6) month old male Wistar rats. Cells were incubated with 500 µM palmitate (37°C, 100% O2) for 4 hrs and stained with 0.5 µM JC-1 or 0.5 µM Rhodamine 123. Δψm was studied using confocal microscopy. Exposure of aged cardiomyocytes to palmitate for 4 hrs resulted in a marked reduction in the JC-1 red fluorescence (figure) and an increase in Rhodamine 123 fluorescence, which indicates a decline in the number of energised mitochondria. In contrast, no significant change in fluorescence was observed in 6 month cells. These results suggest that palmitate can adversely affect mitochondrial function in the ageing heart and that this may be an important contributory factor to the development of heart failure.


Graphic
Figure 3

041 ENDOTHELIAL PROGENITOR CELLS ARE FOUND IN RESOLVING VENOUS THROMBUS

B. Modarai*, B. Sawyer, K. G. Burnand, A. Smith.Academic Department of Surgery, King’s College, London SE1 7EH

This study aimed to determine whether recanalisation during venous thrombus resolution depends on angiogenesis or de novo neovascularisation from circulating endothelial progenitors.

Irradiated mice were reconstituted with bone marrow from transgenic donors expressing green fluorescent protein (GFP) linked to the Tie2 promoter. Thrombi were formed in 2 groups of 6 mice. GFP expressing cells were located in sections of thrombi taken after 7 and 14 days. Fluorescent cell numbers were quantified and GFP expression co-localised with the macrophage marker, MAC3. FACS analysis, using the anti-endothelial antibodies, CD34 and VEGFR2, was also carried out on blood from 3 cohorts of wild-type animals that had either a thrombus induced (n = 18), a sham operation (n = 18), or no operation (n = 10).

Sections of thrombus from all transplanted animals contained GFP expressing cells around the perimeter of the thrombus at day 7. By day 14 the cells were found throughout the thrombus and their number had increased by 3-fold (p = 0.0022). No GFP expressing cells were found lining the new vascular channels that formed at either time interval. Approximately two thirds of the GFP expressing cells also expressed MAC3. Almost twice as many circulating CD34+/VEGFR2+ cells were found at day 3 in animals with thrombus compared with sham controls (p = 0.0459).

Bone marrow-derived, Tie2 expressing, cells are recruited into the thrombus during resolution. These cells did not appear, however, to line the new channels in the thrombus. The cells also expressed a macrophage phenotype and may represent a population of plastic stem cells that orchestrate thrombus recanalisation.

Abstract 017

Abstract 018

Abstract 023

Abstract 027

Abstract 034. Effect of MMP knockout on plaque characteristics (n = 24 per group and all differences p<0.05)

Abstract 035

Abstract 038

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001 RE-EXPRESSION OF A CONSTITUTIVELY-ACTIVE CYCLIN B1:CDC2 COMPLEX IN ADULT RAT CARDIOMYOCYTES IS SUFFICIENT TO REINITIATE CELL DIVISION

K. A. Bicknell*, G. Brooks.School of Pharmacy, The University of Reading, Reading Berskhire RG6 6AJ

The ability of the myocardium to repair itself following a myocardial infarction is severely impaired by the limited capacity of adult ventricular cardiomyocytes to divide. Consequently, the damaged region of the myocardium is replaced by scar tissue and cardiac function is often irreversibly impaired. We previously have shown that adult cardiomyocytes normally do not divide as a consequence of cell cycle arrest in G0/G1 and G2/M. However, during pressure overload-induced hypertrophy, a partial reactivation of the cell cycle machinery occurs that permits adult cardiomyocytes to progress through the G1/S transition prior to their accumulation in G2/M. Interestingly, expression and activity of the G2/M phase cyclin:CDK complex, cyclin B1:CDC2, is not detected in normal adult cardiomyocytes nor in cardiomyocytes undergoing hypertrophy. Thus, we hypothesised that expression and activity of cyclin B1:CDC2 might limit adult cardiomyocyte proliferation. We have used recombinant adenoviruses to re-express cyclin B1 and/or a constitutively active form of CDC2, CDC2AF, in isolated adult rat cardiomyocytes. Re-introduction of cyclin B1 or CDC2AF alone did not result in an increase in cyclin B1:CDC2 kinase activity nor proliferation in adult myocytes compared to control cultures. However, cyclin B1:CDC2 kinase activity was increased 2.9±0.2-fold in cyclin B1 and CDC2AF co-infected cultures compared to controls. Furthermore, re-expression of both cyclin B1 and CDC2AF in adult cardiomyocytes resulted in a significant increase in the number of tropomyosin-positive myocytes (144%±16%), 24 hours after infection, compared to control cultures (100%±8%, n = 5, p<0.05). Interestingly, an increased number of smaller mononuclear tropomyosin-positive myocytes was observed in cyclin B1 and CDC2AF co-infected cultures (17.5%), compared to controls (7.1%). In this study, we have shown that over-expression of the cyclin B1:CDC2 complex reinitiates cell division in adult cardiomyocytes in vitro. Thus, therapeutic strategies targeting the cyclin B1:CDC2 complex might reinitiate cell division in mature cardiomyocytes in vivo and facilitate myocardial regeneration following injury.

002 A GP91PHOX-CONTAINING NADPH OXIDASE CONTRIBUTES TO THE CONTRACTILE DYSFUNCTION ASSOCIATED WITH PRESSURE-OVERLOAD CARDIAC HYPERTROPHY

D. J. Grieve*, J. A. Byrne, J. Layland, A. C. Cave, A. M. Shah.Cardiovascular Division, King’s College London

Reactive oxygen species (ROS) generated by NADPH oxidases may be involved in the pathophysiology of left ventricular hypertrophy (LVH). However, we recently reported that a gp91phox-containing NADPH oxidase is not essential for LVH in response to chronic pressure-overload. Here, we investigated the role of gp91phox in the development of the associated contractile dysfunction. The advent of microconductance technology has significantly advanced the haemodynamic assessment of gene-modified models of cardiovascular disease. In this study, we describe the application of microconductance analysis of cardiac function to the isolated murine ejecting heart. This ex vivo model is complementary to the previously described in vivo preparation, allows assessment without the potentially confounding effects of anaesthetic or neurohumoral influences, and enables careful control of cardiac loading (particularly preload). We first demonstrated that ex vivo pressure-volume relations in the isolated murine heart (n = 8) are sensitive to changes in myocardial contractility induced by aortic constriction. Comprehensive analysis of cardiac pressure-volume relations (1.4 F Millar microconductance catheter) in isolated ejecting hearts demonstrated significant contractile dysfunction in wild-type (WT) bands vs shams in both the steady-state (LV dP/dtmax: 4941±180 vs 5914±211 mmHg/s; stroke work (SW): 12.7±0.8 vs 18.2±1.1 mmHg.μl/mg; P<0.05) and under variable loading conditions (ESPVR: 3.5±0.3 vs 4.8±0.4; EDPVR: 0.080±0.005 vs 0.064±0.005; P<0.05). However, LV contractile dysfunction was preserved in banded gp91phox-/- mice (eg, SW: 17.7±1.5 vs 19.1±1.0 mmHg.μl/mg; ESPVR: 4.7±0.2 vs 4.7±0.3 mmHg; P = NS). Isolated myocyte contractility (% cell shortening) was also significantly decreased in WT bands (4.4±0.3 vs 5.5±0.3%; P<0.05) but not gp91phox-/- bands (4.5±0.3 vs 4.8±0.2%; P = NS).

Conclusions: Although a gp91phox-containing NADPH oxidase is not required for the development of pressure-overload LVH per se, it contributes to the associated contractile dysfunction via changes which are intrinsic to the cardiac myocyte.

003 ENDOTHELIAL TETRAHYDROBIOPTERIN REGULATES ENOS COUPLING/UNCOUPLING INDEPENDENT OF VASCULAR DISEASE: INSIGHTS FROM TARGETED TRANSGENIC MODELS

J. K. Bendall*, N. J. Alp, T. K. Nicoli, K. A. Rockett, S. Kawashima$, M. Yokoyama$, K. M. Channon.Department of Cardiovascular Medicine, University of Oxford, UK; $The First Department of Internal Medicine, Kobe University, Japan

Introduction: Endothelial dysfunction in vascular disease states is associated with eNOS uncoupling so that O2- is produced instead of NO, possibly due to mismatch between eNOS protein and its cofactor tetrahydrobiopterin (BH4). However, the mechanistic relationship between BH4 availability and eNOS activity remains undefined. In particular, whether relative BH4 deficiency alone is sufficient to uncouple eNOS remains unclear.

Methods and Results: We investigated the stoichiometry of BH4-eNOS interactions in vivo by crossing endothelial-targeted eNOS Tg mice (eNOS-Tg) with mice over-expressing endothelial GTP cyclohydrolase (GCH-Tg), the rate limiting enzyme in BH4 synthesis, in which endothelial BH4 levels are increased 3-fold. eNOS protein was increased 8-fold in eNOS-Tg and eNOS/GCH-Tg mice compared with WT. NO synthesis (arginine to citrulline conversion) in lung and aorta, was significantly elevated by 2-fold in eNOS-Tg mice but by 4-fold in eNOS/GCH-Tg mice compared with WT (P<0.05 vs eNOS-Tg). Aortic BH4 levels were depleted 50% in eNOS-Tg compared with WT mice (P<0.05), suggesting oxidative loss of BH4, but BH4 levels were maintained in eNOS/GCH-Tg mice. Endothelial O2- production (oxidative confocal microtopography) was increased 2.5 fold in eNOS-Tg compared with WT aortas but was normalized by L-NAME, suggesting O2- production by uncoupled eNOS. However, in eNOS/GCH-Tg aortas endothelial O2- production was similar to WT, and L-NAME had no effect, indicating preserved eNOS coupling.

Conclusions: These data indicate that eNOS coupling is exquisitely dependent on BH4 levels even in physiological conditions. Thus, strategies to increase eNOS protein without a concomitant increase in endothelial BH4 levels may lead to eNOS uncoupling and paradoxically exacerbate oxidative stress.

004 HYPERTENSION AND ENDOTHELIAL DYSFUNCTION IN MICE FEATURING ENDOTHELIAL CELL-SPECIFIC KNOCKOUT OF ENDOTHELIN B RECEPTORS

A. J. Bagnall*, N. F. Kelland, F. Gulliver-Sloan, G. A. Gray, Y. V. Kotelevtsev, D. J. Webb.Centre for Cardiovascular Science, University of Edinburgh, Scotland. EH8 9XD

Introduction and aims: Endothelin-1 (ET-1) exerts its cardiovascular effects through ETA and ETB receptors (ETBR). Responses to ETBR activation include vasodilatation, vasoconstriction, inhibition of renal sodium reabsorption and clearance of ET-1. Each response may independently modulate blood pressure and blood flow, complicating the interpretation of in vivo pharmacological studies of ETB receptor function. To investigate the contribution of endothelial cell (EC) ETBR to the regulation of blood pressure and vascular tone we have generated mice in which ETBR expression may be spatially regulated.

Methods: Mice featuring loxP sites flanking the ETBR gene (floxed ETBR) were generated by standard gene targeting techniques. Floxed ETBR mice were crossed with Tie2-Cre mice to produce EC-specific ETBR knockout (Flox/Flox Tie2). EC ETBR binding of [125I] ET-1 was measured and wire myography performed to assess endothelial function. Blood pressure (BP) was measured under conscious, unrestrained conditions via in-dwelling catheters in 8–16 week old mice fed with either standard (0.76% NaCl) or high (7.6% NaCl) salt diet. Plasma ET-1 was measured by radioimmunoassay. Wild type (W/W) and single transgenic littermates were used as controls.

Results: EC ET-1 binding was decreased by ∼80% in EC-specific ETBR knockouts (counts/minute/50 μg membrane protein; Flox/Flox Tie2, 581±67; W/W, 3175±268; p<0.001; n = 3). Smooth muscle ETBR-mediated tracheal constriction did not differ between groups. BP was increased in Flox/Flox Tie2 mice (MAP 137.2±6.4 mmHg (n = 5); W/W -/-, 113.7±4.7 mmHg (n = 6; p<0.05) but was unaffected by dietary salt. Plasma ET-1 was increased 4 fold in EC-specific knockouts ([ET-1] pg/ml; Flox/Flox Tie2 12.40±2.95; W/W -/- 2.94±0.83; n = 6;p<0.001). Endothelium-dependent vasodilatation, but not endothelium-independent vasodilatation was impaired following EC ETBR knockout.

Conclusions: Endogenous EC ETBR activation exerts a hypotensive effect that is independent of dietary salt intake and is important for normal endothelial function.

005 THE ROLE OF THE CYCLIN-DEPENDENT KINASE INHIBITOR, P27, IN CARDIAC MYOCYTE HYPERTROPHIC GROWTH

I. C. Kavanagh*, K. A. Bicknell, G. Brooks.School of Pharmacy, The University of Reading, Reading, Berkshire. RG6 6AJ

In order to compensate for increased workload, cardiac myocytes undergo hypertrophic growth to increase their cell size, since they have a limited capacity to divide. At the molecular level, hypertrophic growth involves a partial re-activation of the cell cycle machinery, enabling the cardiac myocyte to advance through the G1/S transition prior to accumulation in G2/M. Cell cycle progression is controlled positively by regulators such as the cyclin-CDK complexes and negatively by regulators such as CDK inhibitors. One such inhibitor is p27 that plays an important role in restricting cell cycle progression at the G1/S border. Previously, we have demonstrated that p27 is down-regulated in response to pressure-overload-induced left ventricular hypertrophy in the rat and, therefore, might play a role in the regulation of hypertrophic growth. To extend our knowledge of how p27 controls hypertrophy, we have treated isolated rat neonatal myocytes with various hypertrophic agonists, including serum and endothelin-1. Hypertrophy was observed with all agonists as confirmed by increases in cell size (microscopy) and the induction of ANF and BNP mRNA expressions. We also demonstrated that the levels of p27 protein were downregulated in neonatal myocytes, in response to these agonists. An ideal model of p27 deficiency is the p27 knockout mouse and we have attempted to use this model to isolate p27-deficient cardiac myocytes in order to study their response to hypertrophic stimuli. However, recent findings from our laboratory and others (Deng et al. 2000. Circulation Research), have demonstrated that wildtype neonatal mouse myocytes undergo autonomous hypertrophy, as determined by increased cell size and total protein levels, when cultured in serum free medium. Therefore, neonatal mouse myocytes are not a good model for studying hypertrophic responses in vitro. Future in vivo studies to compare the effect of pressure-overload-induced hypertrophy between p27+/+ and p27-/- mice, will identify whether myocytes from both backgrounds respond similarly to hypertrophic stimuli.

006 ROLE OF A GP91-CONTAINING NADPH OXIDASE IN CARDIAC MYOCYTE HYPERTROPHY INDUCED BY G-PROTEIN COUPLED RECEPTOR AGONISTS

A. L. Kho*, M. Zhang, A. M. Shah, A. C. Cave.Cardiovascular Division, King’s College London

Although recent studies suggest a critical role for NADPH oxidase-derived reactive oxygen species (ROS) in the pathogenesis of angiotensin-II induced cardiac hypertrophy, the role of the NADPH oxidase in other G-protein coupled receptor (GPCR) agonist-induced hypertrophy is unclear. Thus we have investigated the signalling pathways leading to ROS production and hypertrophy in isolated neonatal rat cardiomyocytes exposed to the GPCR agonists endothelin-1 (ET1, 10 nM) or phenylephrine (PE, 10 μM) or vehicle (serum-free control, SF). Hypertrophy was assessed by cell size and quantitative RT-PCR of atrial natriuretic factor (ANF) mRNA, normalised by β-actin. NADPH oxidase activity was assessed by lucigenin (5 μM) chemiluminescence and in situ ROS production by dihydrochlorofluorescein (DCF) fluorescence.

24 h exposure to PE or ET1 increased cell size and ANF mRNA levels (PE: 13.8±3.4 fold increase vs SF; ET1: 5.3±0.6 fold increase vs SF; P<0.05) concomitant with an increase in NADPH oxidase-dependent ROS production (PE: 90±18%; ET1: 118±21%; both P<0.05 vs SF) and DCF fluorescence (133±41% and 182±49% respectively; P<0.05). The PKC inhibitor, bisindolylomaleimide (Bis, 5 μM), significantly inhibited both PE and ET1-induced NADPH oxidase activation (PE+Bis:17±20%; ET1+Bis: 12±13%; P<0.05 vs PE/ET1 alone), but not PE or ET1 induced increases in ANF mRNA (PE+Bis: 14.3±5.2; ET1+Bis: 13.7±3.1-fold increase, P = NS vs PE/ET1 alone). However, the antioxidant, BHA (butylated hydroxyanisole: 50 μM) significantly reduced PE-induced rises in ANF mRNA (PE+BHA: 3.4±0.6, ET1+BHA: 2.8±1.3; both P<0.05 vs PE/ET1 alone). Transfection with a gp91phox antisense (AS) oligonucleotide had no effect on either PE or ET1-induced DCF fluorescence (PE+AS, 95±22% and ET1+AS 116±20% of the PE/ET1 response alone).

PE and ET1 induce a PKC-dependent activation of NADPH oxidase in myocytes, which does not appear to involve Nox 2. The PE/ET1 induced increase in ANF mRNA is dependent on ROS production, implicating a redox-sensitive signalling pathway. However, failure to inhibit these responses with Bis suggests there are multiple pathways to ANF induction.

007 GLYCATED ALBUMIN STIMULATES OXIDANT STRESS IN CULTURED NEONATAL RAT CARDIAC MYOCYTES: INVOLVEMENT OF GP91PHOX-CONTAINING NADPH OXIDASE

M. Zhang*, A. L. Kho, A. M. Shah, A. C. Cave.Cardiovascular Division, King’s College London, London SE5 9PJ

Background: Nonenzymatic glycation is increased in diabetes. Although several studies have implicated an important role for advanced glycation end products in many of the complications of diabetes, the effects of early-glycation Amadori-modified proteins, such as glycated serum albumin (GSA), remain less well defined. However, in vascular smooth muscle cells, GSA resulted in a significant activation of redox sensitive signaling molecules and a significant stimulation of growth and migration. Thus the aim of this study was to investigate the effects of GSA on reactive oxygen species (ROS) production by cardiomyocytes and to define the underlying enzymatic source(s) and signalling pathways involved.

Methods and Results: Cultured neonatal rat myocytes were incubated with GSA or vehicle (bovine serum albumin, BSA) for up to 24 h. GSA dose-dependently increased in situ ROS production, assessed by dihydrochlorofluorescein fluorescence, with a optimum effect at 400 µg/ml (152±10% versus BSA (100%) at 24 h, P<0.01; n = 3). The earliest significant increase in ROS occurred after 2 h of incubation (128±1%) reaching a maximum at 24 h. Similarly NADPH-dependent O2- generation in cell homogenates was significantly increased at 24 h (179±15% vs BSA (100%); P<0.01, n = 3). ROS production was inhibited by the NADPH oxidase inhibitors apocycin (500 µM) or diphenyleneiodonium (DPI 5 µM), by Tiron (20 mM), but not by L-NAME (100 µM) or rotenone (10 µM). Transfection with gp91phox antisense oligonucleotides significantly reduced GSA-induced in situ ROS production at 24 h (61.4±8.3% decrease, n = 3, P<0.01). The PKC inhibitor, bisindolylmaleimide I (Bis, 5 µM) significantly inhibited AGE-induced NADPH oxidase activation at 24 h (AGE+Bis:120±16 vs AGE:179±15% of control; P<0.05; n = 3). The AGE-induced increase in ROS at 24 h was accompanied by nuclear translocation of NF-κB (inhibited by N-acetylcysteine (NAC)) and an increase in ANF mRNA expression (2.76±0.48 by real-time RTPCR) normalised to BSA control (P<0.05). NAC and apocynin significantly inhibited this increase in ANF mRNA expression (0.96±0.38 and 1.01±0.84 respectively; P<0.05).

Conclusion: GSA stimulates cardiomyocyte ROS production through PKC-dependent activation of a gp91phox-containing NADPH oxidase. The increase in ROS is associated with NF-κB activation and upregulation of ANF mRNA. These findings suggest that early glycated proteins may contribute to cardiac myocyte dysfunction in diabetes.

008 EXOGENOUS TISSUE FACTOR SUPRESSES THE EXPRESSION OF ATRIAL NATRIURETIC FACTOR IN H9c2 RAT CARDIOMYOCYTES

G. A. Frentzou*, C. Ettelaie, A-ML. Seymour.Department of Biological Sciences, University of Hull, Cottingham Rd., Hull, HU6 7RX

The adaptive response of the heart to chronic mechanical overload, termed “cardiac hypertrophy” is an important risk factor in the development of heart failure. During the onset of cardiac hypertrophy, tissue factor (TF), the initiator of the extrinsic pathway of blood coagulation, is shown to be upregulated in the myocardium. We investigated the influence of different concentrations of exogenous human recombinant TF (5 nM, 50 nM, 500 nM and 2 µM) on the expression of atrial natriuretic factor (ANF) in H9c2 rat cardiomyocytes over a period of 15 days, in the presence of 10% foetal calf serum (FCS). The cells were harvested at 1, 2, 5, 10, and 15 days, and analysis of ANF expression carried out by semi-quantitive RT-PCR and western blotting. The ANF expression peaked on the first day (3 and 5 folds for mRNA and protein respectively) and was subsequently suppressed (down to 20% of the control) over the period of investigation. Our data indicate that exogenous TF either alone, or in combination with serum factors, is capable of inhibiting the expression of ANF. This suggests that TF may play a crucial role in cellular homeostasis.

“The support of National Heart Research Funds is acknowledged”

009 UP-REGULATION OF UROKINASE (uPA) MAKES MONOCYTES HIGHLY FIBRINOLYTIC

J. Humphries*, J. Gossage, B. Sawyer, K. Burnand, A. Smith.Academic Department of Surgery, Kings College London, SE1 7EH

Background: The balance between uPA and plasminogen activator inhibitors (PAI) may determine the fibrinolytic balance of the monocyte. The aim of this study was to over-express uPA and determine the effect on monocyte fibrinolytic activity.

Methods: Monocytes were isolated from 8 normal individuals and pre-treated with macrophage colony stimulating factor.These cells were then transfected with 1000 multiplicity of infection (MOI) of uPA expressing adenovirus (ad-uPA) or a blank adenovirus control (ad-0). Cells were harvested 4 days post transfection. Cell conditioned media were analysed for uPA, tissue-type plasminogen activator inhibitor (tPA), urokinase plasminogen activator receptor (uPAR), PAI-1, and PAI-2 using ELISA assays. uPA activity was also estimated by quantifying zones of lysis on a fibrin plate. Total soluble protein and cell viability were assessed using colorimetric assays.

Results: Transfection of monocytes with ad-uPA raised median uPA antigen production by 400 fold compared to zero uPA production in all ad-0 treated controls (p = 0.03). A 160 fold increase in median uPA activity, as determined by fibrin plate lysis, was observed in cells with ad-uPA. No uPA activity was measured in any ad-0 treated cells (p<0.0001). No tPA could be measured in monocytes irrespective of treatment. Up-regulation of uPA decreased PAI-2 production (median 448 (range 140–787) ng/mg soluble protein) compared to controls (median 1000 (range 135–1000) (p = 0.06)). Treatment with ad-uPA also resulted in a significant increase in monocyte uPAR production (p = 0.03) and a significant decrease in PAI-1 production (p = 0.03) when compared to ad-0 treated controls. Adenoviral transfection did not affect cell viability.

Conclusion: Treatment of monocytes with ad-uPA increased monocyte uPA and uPAR production and also decreased production of its main inhibitors. These fibrinolytic cells are being infused into models of deep vein thrombosis to ascertain whether they might accelerate thrombus resolution.

010 ALTERNATIVE SPLICING OF SERUM RESPONSE FACTOR

A. R. Gordon*, J. Metcalfe, P. R. Kemp.Department of Biochemistry, University of Cambridge, Cambridge

Serum response factor (SRF) is a transcription factor that controls smooth muscle specific gene expression and plays a key role in determining the phenotype of smooth muscle cells (SMC). Alteration in the differentiated state of SMC is a common feature of many cardiovascular diseases and changes in SRF expression have recently been implicated in the development of cardiovascular disease. Overexpression of SRF or of a mutant inhibitory form of SRF in mice resulted in increased heart to body weight ratios and bi-ventricular enlargement. Furthermore, recent studies have shown that SRF is alternatively spliced into four isoforms (SRF-L, SRF-M, SRF-S & SRF-I) and that the pattern of SRF splicing is altered in heart failure. The aim of this study is to investigate the effect of SRF splicing on SRF activity and to determine the role of SRF splicing in the control of cell phenotype during normal cardiovascular development and disease.

C2C12 myoblasts and P19 embryonic carcinoma cells were transfected with SRF-L and SRF-M. SRF-M, which lacks part of the transactivation domain, had reduced activity compared to the full length SRF-L. Furthermore, the expression of SRF-M compared to SRF-L in these cells was correlative with the expression of muscle-specific genes. Thus, the relative proportions and activities of SRF-L and SRF-M may have a significant role in the control of muscle-specific gene expression.

To investigate this further, we have generated targeting constructs which will be used to generate mice that are unable to splice SRF into isoforms SRF-L and SRF-M and will therefore lack expression of these proteins. As well as global knockout of SRF-L, we have floxed the gene which will allow for the tissue-specific deletion of SRF-L. Tissue specificity will be achieved by crossing mice with SM-MHC-Cre and MLC2v-Cre mice to generate smooth muscle and cardiac specific knockouts respectively. Analysis of mice will determine if lack of specific SRF isoforms contributes to an abnormal cardiovascular phenotype.

011 THE EFFECT OF TUMOUR NECROSIS FACTOR α INFUSION BY IMPLANTABLE PUMP ON MURINE CARDIAC FUNCTION

M. E. Faircloth*, J. E. Clark, K. Dighe, M. S. Marber.Cardiovascular Division, KCL, Rayne Institute, St Thomas’ Hospital, London SE1 7EH

Introduction: Elevated concentrations of Tumour Necrosis Factor α (TNFα) occur in chronic heart failure, and are associated with poor left ventricular (LV) function and outcome. In mice, cardiac restricted TNF expression leads to chamber dilatation but it is not known whether TNF infusion results in a similar phenotype.

Aims: We have assessed the LV characteristics in mice infused for seven days with TNFα or diluent.

Methods: Osmotic infusion pumps were implanted subcutaneously in 12 male C57B6 mice. Group 1 (n = 6, weight 26.2 g) received recombinant murine TNFα at a rate of 40 or 80 µg/kg/min, and group 2 (n = 6, weight 28.3 g) received 0.9% saline. After seven days, cardiac function in the two groups was assessed by the use of a miniature conductance catheter passed into the LV via the carotid artery, crossing the aortic valve.

Results: No differences were observed between groups for mouse weight, cardiac output (CO), ejection fraction (EF), stroke work, or end-diastolic pressure. There was a slight increase in heart rate (HR) in the TNF group (p<0.05), in which was also observed a marked increase in end-systolic and end-diastolic volumes (p<0.01 for both), and a reduction in end-systolic pressure, dP/dtmax and dP/dtmin (p<0.05 for all). A trend was observed towards a reduction in the maximal elastance and end systolic and diastolic elastance.

Conclusions: This first in vivo mouse study of the effects of prolonged TNF infusion demonstrates LV dilatation. The maximum and minimum rates of change in pressure, measures of contractile function, are reduced. These are characteristic changes of the failing heart, and support the hypothesis that TNF is involved in the causation of heart failure, rather than rising in response to it, and this is supported by the change in the elastance. That the SV, CO and EF have not been seen to be different between groups perhaps reflects early, partially compensated heart failure, a theory further supported by the slight rise in heart rate.

012 P38-MAPK MEDIATES THE EARLY NEGATIVE INOTROPIC EFFECT OF TUMOR NECROSIS FACTOR-α. EVIDENCE OF SYNERGY BETWEEN A DIRECT NEGATIVE INOTROPIC EFFECT AND CORONARY CONSTRICTION

M. Bellahcene*, X. B. Cao, J. Layland, M. Tanno, A. M. Kabir, R. S. Haworth, M. Avkiran, M. S. Marber.Cardiovascular Division, KCL, The Rayne Institute, St Thomas’ Hospital, London SE1 7EH, UK

Background and goal: Tumor necrosis factor-α (TNF) exerts a negative inotropic effect on the myocardium from various species. TNF activates p38-MAPK and this kinase is thought to depress contractility in a calcium-independent manner. We therefore examined TNF effects on contractility in mice lacking the p38-MAPK activator, MKK3.

Methods: The left ventricular developed pressure (LVDP; isovolumic contraction), coronary flow, p38-MAPK and HSP27 phosphorylation, as well as the end-diastolic v LVDP (or Frank-Starling) relationship were analyzed in C57BL/6 outbred, mkk3 wild-type (WT), and mkk3 knock-out (KO) isolated mouse hearts exposed to 10 ng/ml TNF i.c. for 15 min after 40 min of stabilization. Some hearts received SB203580 (p38-MAPK catalytic site inhibitor; 1 μM) for 20 min starting 5 min before TNF infusion. All protocols were run under constant pressure and constant flow conditions since TNF also has vasoconstrictive properties.

Results: TNF (10 ng/ml) for 15 min significantly reduced LVDP and coronary flow in outbred and mkk3-WT mice. This early negative inotropic effect of TNF was associated with a significant phosphorylation of both p38-MAPK and HSP27. However, TNF did not reduce the LVDP and did not phosphorylate p38-MAPK and HSP27 in mkk3-KO mice despite some reduction in coronary flow. Similarly, TNF caused a significant depression of the Frank-Starling relationship in both outbred and mkk3-WT, but not mkk3-KO mice. Furthermore, SB203580 attenuated TNF-induced negative inotropy, as well as p38-MAPK and HSP27 phosphorylations. Comparing constant pressure vs constant flow models of heart perfusion, we found that TNF-induced coronary constriction can act in synergy with a direct effect of TNF on cardiomyocytes to cause p38-MAPK activation, and consequent negative inotropy.

Conclusion: Our results implicate p38-MAPK in the early and dual negative inotropic effect of TNF, and may be of relevance to the pathogenic actions of this cytokine in heart failure.

013 PROTECTION AGAINST ENDOTOXEMIA-INDUCED CONTRACTILE DYSFUNCTION IN MICE WITH CARDIAC-SPECIFIC EXPRESSION OF SLOW SKELETAL TROPONIN I

J. Layland*, A. C. Cave, D. J. Grieve, E. Sparks, J. C. Kentish, R. J. Solaro, A. M. Shah.Cardiovascular Division, King’s College London and University of Illinois at Chicago

The intrinsic impairment of cardiomyocyte contractility characteristic of gram negative endotoxaemia has been partly attributed to a reduction in myofilament Ca2+-responsiveness and has also been associated with increased cardiac troponin I (cTnI) phosphorylation at serines 23 and 24 (residues required for the protein kinase A (PKA)-dependent reduction of myofilament Ca2+-sensitivity following β-adrenoceptor stimulation). To investigate the functional significance of increased TnI phosphorylation in endotoxemia, we studied the contractile effects of systemic bacterial lipopolysaccharide (LPS) treatment in transgenic mice (TG) with cardiac-specific replacement of cTnI by slow skeletal TnI (ssTnI, lacking PKA phosphorylation sites) and matched non-transgenic littermates (NTG) on a CD1 background. In wild-type CD1 mice, LPS treatment (6 mg/kg, IP, 16-18 hours) caused a 37% reduction in isolated myocyte unloaded sarcomere shortening from 6.1±0.2 to 3.9±0.2% (1 Hz, 32°C, P<0.05). Similarly, in NTG myocytes, endotoxemia reduced myocyte shortening by 42%, from 6.7±0.2% to 3.9±0.1% (P<0.05) with no change in intracellular Ca2+ transients. However, in the TG group, LPS reduced myocyte shortening by only 13% from 7.5±0.2% to 6.5±0.2% (P<0.05). LPS treatment significantly reduced contractility during β-adrenergic stimulation (10 nM isoprenaline) in NTG myocytes but not in TG myocytes, even though isoprenaline-induced increases in Ca2+ transient amplitude were similar in both groups. Investigation of the sarcomere shortening-Ca2+ relationship in Triton-skinned cardiomyocytes revealed a significant reduction in myofilament Ca2+-sensitivity following LPS treatment in NTG myocytes, an effect that was substantially attenuated in TG myocytes. In conclusion, the replacement of cTnI with ssTnI in the heart provides significant protection against endotoxemia-induced cardiac contractile dysfunction, most probably by preserving myofilament Ca2+-responsiveness by preventing TnI phosphorylation of PKA-sensitive sites.

014 NO ROLE FOR CALCINEURIN IN SENSITIVITY TO ISCHEMIA/REPERFUSION, MAPK/SAPK ACTIVATION OR ISCHEMIC PRECONDITIONING IN THE EX VIVO MOUSE HEART

K. Obasanjo-Blackshire*, O. Beuno†, J. Molkentin†, M. Marber, R. Heads.Cardiovascular Division, KCL, UK and †Children’s Hospital Medical Center, Cinncinnati, USA

The Ca2+-dependent phosphatase calcineurin A (CnA) plays an important role in inflammation, protection and injury in cardiomyocytes, embryonal heart development and in the development of hypertrophy and heart failure in adults. The developmental and hypertrophic effects of CnA are partly mediated at the transcriptional level via regulation of the NFAT family of transcription factors. CnA may also modulate the activities of several signal transduction pathways including MAPKs and SAPKs. In this study we have used hearts from wild type (WT) mice or mice with targeted disruption (KO) of the CnAβ gene (the major cardiac isoform) to determine differences in sensitivity to ischemia/reperfusion (I/R), in MAPK/SAPK activation and in ischemic preconditioning (IPC) which may be attributable to CnAβ. Intraperitoneal injection of LPS into mice 24 hours prior to removal of the heart and analysis of iNOS expression revealed robust iNOS expression in WT hearts which was abolished in CnAβ KO hearts, suggesting a role for CnAβ in inflammatory signalling. Hearts from outbred C57BL6 mice subjected to 45 min global ischemia (I) and 90 min reperfusion (R) in Langendorff mode showed biphasic transient activation of p38-MAPK, during both I and R, whereas both p42/p44-MAPK and JNK (SAPK1) were activated by reperfusion only. No differences in this pattern of MAPK/SAPK activation were seen in either WT or CnAβ KO hearts. There were also no differences in haemodynamic parameters either at baseline or during I/R, or in infarct size between WT and CnAβ KO hearts. The development of acute IPC in response to 6 cycles of 4 min I/6 min R prior to index I/R was also unaffected (WT 17.3±2.2%* v 31.9±4.3% and KO 14.2±1.5%* v 32±3.2%; *p<0.05 vs I/R by ANOVA). These results suggest that despite an effect on the expression of inflammatory mediators in vivo, CnAβ does not play a role in the sensitivity to ischemia/reperfusion, MAPK/SAPK activation or acute IPC in the buffer perfused heart ex vivo.

015 ARE ELAFIN OVEREXPRESSING MICE PROTECTED FROM ACUTE MYOCARDIAL ISCHAEMIA/REPERFUSION INJURY?

J. Sharif., M. Sallenave, G. A. Gray.Centre for Cardiovascular Science; *Centre for Inflammation Research, College of Medicine and Veterinary Medicine, University of Edinburgh. Edinburgh EH8 9JZ

It has been suggested that early local activation of neutrophil derived serine elastases may play an important role in later myocardial remodelling. Recent studies have demonstrated that overexpression of the serine elastase inhibitor elafin improves myocardial function in models of viral myocarditis (Zaidi et al., 1999) and chronic myocardial infarction (Ohta et al., 2004).

As neutrophils play a major role in cardiac reperfusion injury, the aim of the present study was to investigate whether mice overexpressing elafin (under control of the preproendothelin promoter, EE mice) would be protected from damage in an acute model of myocardial ischaemia-reperfusion.

11-13 week old male C57Bl6 or EE mice (backcrossed onto a C%&Bl6 background) were anaesthetised (ketamine/xylazine, i.p.) and instrumented for measurement of blood pressure. Tracheotomy was performed to permit artificial ventilation. The left coronary artery was ligated for 45 minutes, followed by 2 hours of reperfusion. The ischaemic area (area at risk, AAR) was assessed by infusion of Evans Blue dye at the end of the experiment and ischaemic damage was later assessed in heart sections using tetrazolium chloride.

In control C57Bl6 mice the AAR was 57±9% of the left ventricle and the necrotic zone comprised 38±6% of the AAR. Neither the AAR (63±3%) nor the necrotic zone (40±6%) were significantly modified in EE mice.

The lack of modification of the necrotic zone in the surrent study, together with the observed lack of change in infarct size in chronically infarcted mice (Ohta et al., 2004) suggest that elafin overexpression may target processes other than those involved in immediate ischaemia related damage in the myocardium.

• Ohta K, et al. Am J Physiol 2004;87(1):H286–92.

• Zaidi SHE, et al. J Clin Invest103:1211–9.

This study was supported by the British Heart Foundation PG/03071

016 HYPERTENSION AND CARDIAC HYPERTROPHY IN BTEB3 NULL MICE

M. Keramatipour, C. A. Goddard, W. H. Colledge, P. R. Kemp*.Department of Biochemistry, University of Cambridge, CB2 1QW

We have previously shown that the Krüppel-like factor BTEB3 is expressed in vascular smooth muscle cells and activates the SM22α promoter in vitro by binding to CG rich sequences in the promoter. Using a similar approach we have also found that BTEB3 like similar KLFs activates the cardiac troponin C promoter but can also act as an inhibitor of gene expression including the SM α-actin promoter. BTEB3 is widely expressed in the mouse embryo with strong expression in the heart at E11.0. To determine the role of BTEB3 in the mouse we have generated mice in which exon 1 of the BTEB3 gene is replaced by a neomycin resistance gene.

Genotyping of 246 live born mice has shown that BTEB3-/- mice are born but are under-represented (by a factor of 50%) in the litters at the time of genotyping. The surviving mice are fertile and the litters produced are of a reasonable size. However, the mice show an increase in the ratio of heart weight to body weight (approximately 20%) both at 3 months and at 6 months regardless of whether they are born to -/- parents or +/− parents. This cardiac hypertrophy is accompanied by an increase in systolic blood pressure of approximately 20 mm Hg. Preliminary analysis of the hearts is consistent with dilatation of the right ventricular chamber.

To determine the mechanisms behind these changes the cardiovascular system we are analysing gene expression in the right and left ventricles and in the aorta. The cause of embryonic lethality is also currently under investigation.

017 IDENTIFICATION OF GENES THAT ARE DIFFERENTIALLY EXPRESSED IN HUMAN HEART FAILURE USING QUANTITATIVE REAL TIME POLYMERASE CHAIN REACTION (QRT-PCR)

A. B. Goulter*, J. C. Allen, K. L. Clark.Pharmagene Laboratories, Royston, Herts, SG8 5HD, UK

The use of sensitive gene expression profiling techniques applied to human tissue can reveal novel information regarding well studied targets. Therefore, we investigated mRNA expression of a range of established pharmacological targets in samples of human left ventricular free wall classified as non-diseased, idiopathic dilated cardiomyopathy (IDC) and ischemic cardiomyopathy (ICM) (see table below). In every PCR reaction, the level of the housekeeping gene, GAPDH was also determined (no differences were observed in expression levels between the three groups). Atrial natriuretic factor (ANF) and ACE mRNA were evaluated as positive controls and confirmed to be significantly up-regulated in IDC and ICM relative to the control group. Histamine H2 receptor (H2) and phosphodiesterase (PDE) IV (isoform 2) mRNA levels were down-regulated by approximately 2-fold in both IDC and ICM groups. PDE III (isoform 2) was down-regulated by 3 and 5-fold in IDC and ICM, respectively. This study shows for the first time in the human heart, levels of mRNA for H2, PDE III-2 and PDE IV-2 are down-regulated in both IDC and ICM versus non-diseased myocardium.

Conclusion: This study demonstrates the capability of QRT-PCR to reveal novel data regarding the transcriptional regulation of existing drug targets in human heart failure. Further target validation is now warranted.

018 ABSENCE OF α7-INTEGRIN LEADS TO PROGRESSIVE STRUCTURAL CHANGES IN THE HEART

R. Nadif-Savey*, N. Ismayilova$, L. Neyses, U. Mayer$, M. Emerson.Division of Cardiology and $Wellcome Trust Centre for Cell-Matrix Research, University of Manchester, Oxford Road M13 9PT

α7β1 is the major laminin-binding integrin in skeletal, heart and smooth muscle. Integrins are essential for normal structural development and signalling in the heart although the role of the α7 subunit remains undefined. We are therefore investigating the role of α7 integrin in heart integrity and function by studying the cardiac phenotype of α7 deficient mice. Gross histological comparisons between wild type (WT) and knock-out (KO) hearts revealed that the α7 deficient hearts had a clearly abnormal structure defined by an elongated vertical-axis and reduced heart weight (195.9±12.4 mg in WT versus 125±7.9 mg* in KO; *P<0.05) at 12 months of age. Echocardiography showed no significant differences in measured parameters at 3 months of age although at 9 months the data was as shown below:

Left ventricular catheterisation of 6 month old mice showed normal heart function in KO animals basally and following β-adrenergic stimulation. We conclude that deletion of the α7 gene causes a novel, age-dependent cardiac phenotype characterised by changes in chamber dimensions and wall thickness. In younger animals, however, haemodynamic performance is not impaired. Further studies are required to fully characterise the functional implications of α7 integrin deletion and to determine the structural and signalling mechanisms through which α7 modifies heart structure and function.

019 REGIONAL CARDIAC ACTIVITIES OF MATRIX METALLOPROTEINASE-2 & -9 FOLLOWING CHRNIC MI IN MICE: THE INFLUENCE OF GENETIC BACKGROUND & OESTROGEN

I. Sharif, S. C. Riley*, R. Leask*, E. Bruce, D. J. Webb, G. A. Gray.Centre for Cardiovascular Science & *Centre for Reproductive Biology, College of Medicine and Veterinary Medicine, University of Edinburgh

Cardiac rupture is a feature of chronic MI in the mouse, that has been linked to activation of metalloproteinases (MMPs) -2 and –9 in the myocardium. We have observed differences in the incidence of rupture in mice with different genetic backgrounds and also a doubling of the incidence of rupture in ovariectomised (ovx) female mice treated with oestrogen (E).

The aim of the present study was to map regional activation of MMP-2 and -9 following MI in the mouse and to investigate the influence of genetic background and E on their activation pattern. 11–13 week old male and ovx females with or without E supplementation underwent coronary artery ligation for induction of MI. At 4 days post-MI, the activity of MMP-2 and -9 were determined by gelatin zymography in the infarcted, border and non-infarcted regions of left ventricle, septum and right ventricle.

Following MI, the activity of both MMPs was most dramatically increased in the infarct and infarct border zones of all hearts studied compared to control mice. In hearts of mixed background mice the increases, particularly in MMP-9 activity were higher compared to those of C57bl/6J hearts. E had no significant influence on the activity of either MMP in control of MI mice.

Enhanced activation of MMP-2 and -9 enzymes in the infarct and border may account for the higher level of cardiac rupture in C57bl/6J-129/SvJ compared to C57bl/6J mice. However, these MMPs are not directly implicated in the increased incidence of cardiac rupture caused by E.

This work is supported by British Heart Foundation (PG/99192 and FS/03114)

020 A GP91-PHOX-CONTAINING NADPH OXIDASE IS CRITICAL FOR ANGIOTENSIN II-INDUCED INTERSTITIAL CARDIAC FIBROSIS

S. Johar*, A. C. Cave, D. J. Grieve, A. M. Shah.Cardiovascular Division, Kings College London

The development of interstitial fibrosis and cardiac hypertrophy may be independently regulated. A gp91phox-containing NADPH oxidase was previously shown to be necessary for development of cardiac hypertrophy in response to subpressor angiotensin II (AngII) infusion. In this study, we investigated its role in AngII-induced interstitial cardiac fibrosis and the contribution of aldosterone to this process. Mice lacking gp91phox (KO) and wild-type mice (WT) received AngII (1.1 mg/kg/day for 14 days) or vehicle via osmotic minipump. A group of WT AngII-infused mice also received spironolactone, an aldosterone antagonist (SPIRO, 200 mg/kg/day) in chow (n⩾6 all groups). AngII induced a similar rise in systolic blood pressure in WT and KO (WT: 107±4 to 161±8 mmHg, P<0.05; KO: 107±2 to 151±5 mmHg, P<0.05) which was unchanged by SPIRO in WT (167±9 mmHg). Interstitial cardiac fibrosis (Massons Trichrome) increased significantly with AngII in WT (7.2±0.7% to 11.5±1.0%; P<0.05) but not in KO (6.0±0.6% to 5.8±1.0%, P = NS). The AngII-induced increase in fibrosis in WT was inhibited by SPIRO (7.5±1.0%). CCCcsdasasd Consistent with these data, expression of fibronectin and procollagen I mRNA increased in AngII-treated WT by 2.9±0.6 and 3.0±0.7 fold respectively (both P<0.05) but not in KO (1.2±0.1 and 1.4±0.3 fold respectively, both P = NS). SPIRO partially inhibited the AII-induced increase in procollagen I (1.9±0.2 fold) but not fibronectin (2.8±0.3 fold) in WT mice. Myocardial NADPH oxidase activity (5 µM lucigenin chemiluminescence) was increased in AngII-treated WT (2.9±0.4 to 5.1±0.4 integrated light units [ILU]; P<0.05) but not with AngII treatment in KO (3.2±0.5 vs 3.5±0.3 ILU; P = NS). Interestingly, SPIRO also inhibited AngII-induced oxidase activity in WT (3.3±0.1 ILU. These data suggest a critical role for a gp91phox-containing NADPH oxidase in AngII-induced interstitial cardiac fibrosis which is independent of blood pressure or hypertrophy per se. At least part of the effect of AngII may be mediated by aldosterone.

021 OVEREXPRESSION OF IGF BINDING PROTEIN-2 IN MICE PROTECTS AGAINST INSULIN RESISTANCE AND OBESITY

S. B. Wheatcroft, P. A. Crossey, M. Modo, S. Williams, J. Miell, V. Ezatt, A. M. Shah, M. T. Kearney*.Cardiovascular Division, King’s College London, UK

Obesity and insulin-resistance are independent risk factors for the development of cardiovascular disease, but the mechanisms underlying these conditions are not fully understood. Insulin-like growth factor (IGF)-I modulates insulin sensitivity and stimulates adipocyte differentiation. IGF binding protein-2 (IGFBP-2) is an important regulator of IGF bioactivity. To test the hypothesis that IGFBP-2 modulates the development of insulin resistance and obesity, we overexpressed IGFBP-2 in transgenic (TG) mice and assessed metabolic parameters in response to aging and high fat feeding. Female TG mice were compared with littermate wild type (WT) controls (n = 6–8 per group). Data are expressed as mean ± SEM.

At 8 weeks of age, fasting glucose and insulin levels, glucose tolerance tests (GTT) and systolic blood pressure (SBP) were similar in TG and WT animals. At 40 weeks, glucose levels were lower in TG mice than WT in a GTT (10.5±0.5 v 12.8±0.4 mmol/L; P<0.05). TG mice were more insulin sensitive than WT controls in an insulin tolerance test. SBP was also lower in TG mice than WT at 40 weeks (123±2 v 134±2 mmHg; P = 0.001). Susceptibility to nutritional obesity was assessed by feeding a high fat diet for 32 weeks. TG mice gained significantly less weight on the diet than WT controls (9.8±2.3 g v 16.8±2.2 g; P<0.05). Peri-gonadal and mesenteric fat depots were smaller in TG than WT animals (3.7±0.8% v 13.5±1.5%;P =  0.002 and 1.0±0.04% v 2.5±0.5%; P = 0.007). Cross sectional fat area, measured by MRI scanning at the level of the kidneys, was also lower in TG mice than WT controls (40±3% v 54±3% of cross sectional area; P = 0.04). Mean fat cell area was lower in TG mice than WT counterparts (285±33 µm2 v 597±18 µm2; P = 0.003).

In summary, overexpression of IGFBP-2 in mice attenuates age-related changes in glucocompetence and blood pressure, and reduces the susceptibility to nutritional obesity. These data demonstrate a novel and important role for IGFBP-2 in metabolic homeostasis. Funded by the British Heart Foundation

022 MITOCHONDRIAL FATTY ACID TRANSPORT GENES ARE DOWN-REGULATED IN CARDIAC HYPERTROPHY

A. S. Akki*, E. Dyer, S. Richardson, A-ML. Seymour.Department of Biological Sciences, University of Hull, Hull, HU6 7RX

Cardiac hypertrophy is a major risk factor in the development of heart failure. It is characterised by cellular remodelling of the heart. Energy provision is modified in the compensated hypertrophied heart, with an enhanced uptake and utilisation of glucose and a consequent reduction in fatty acid oxidation. The aim of this study was to investigate alterations in gene expression of key enzymes involved in mitochondrial fatty acid transport at an early stage of cardiac hypertrophy using an aortic constriction model. Pressure overload hypertrophy was surgically induced in male Sprague-Dawley rats by abdominal aortic constriction. RNA was extracted from control and hypertrophied hearts 9 weeks post-surgery and gene expression probed using RT-PCR and semi-quantitative analysis. The heart weight to tibial length (Hw/T) ratio was used as an index of hypertrophy. Hw/T ratio was 0.37±0.02 in the control animals. In aortic constriction animals with Hw/T ratios ⩽ 0.38, cardiac expression of Peroxisome proliferators-activated receptor α (PPARα) and Carnitine Palmitoyl Transferase1 (CPT1) were up-regulated relative to Glyceraldehyde phosphate dehydrogenase (GAPDH) and Calsequestrin2 whilst that of Carnitine acylcarnitinetranslocase (CAT) was down-regulated. However, in aortic constriction animals with a greater degree of hypertrophy (Hw/T ratios ⩾0.4), expression of PPARα and CPT1 in hearts were reduced and that of CAT enhanced. These results indicate that PPARα expression modulates expression of CPT1 and CAT in an inverse manner. With increasing hypertrophy, changes in expression of PPARα, CPT1 and CAT occur in parallel with a shift from the beneficial phase of compensated hypertrophy to a maladaptive phase. Thus changes in fatty acid metabolism may contribute to the development of heart failure.

023 EFFECT OF CHRONIC CARNITINE SUPPLEMENTATION ON MYOCARDIAL METABOLISM AND FUNCTION IN EXPERIMENTAL URAEMIA

V. Reddy*, S. Bhandari, A-ML. Seymour.Dept. of Biological Sciences, University of Hull, HU6 7RX

Cardiac complications are the leading cause of mortality in patients with chronic renal failure. Secondary carnitine deficiency frequently occurs in haemodialysis patients and can lead to impaired fatty acid oxidation in the myocardium. In addition, carnitine deficiency is associated with cardiac hypertrophy and heart failure. The aim of this study was to determine the impact of chronic carnitine supplementation on cardiac contractile function and oxidative fluxes in experimental uraemia using 13C NMR spectroscopy. Uraemia was induced in male Sprague-Dawley rats via a two-stage 5/6 nephrectomy. L-Carnitine was administered (5 mM, 0.25 µL/hr) continuously via subcutaneous mini-osmotic pumps for 6 weeks post surgery. Isolated hearts were perfused in the isovolumic mode, with simultaneous monitoring of cardiac function and myocardial oxygen consumption. Carnitine supplementation resulted in a significant rise in serum free carnitine concentration in control (55.4%) and uraemic (77.6%) groups (see table). A significant reduction in heart weight: tibia length ratio and decrease in glucose oxidation was observed following carnitine treatment in control and uraemic groups. In conclusion, chronic carnitine supplementation results in a reduction in left ventricular hypertrophy, associated with a switch in substrate selection from glucose to alternate fuels.

024 INVESTIGATION OF THE CLEARANCE OF ENDOTHELIN –1 FROM PLASMA USING A MURINE ENDOTHELIAL CELL SPECIFIC KNOCKOUT MODEL

N. F. Kelland*1, A. J. Bagnall1, F. H. Gulliver-Sloan1, R. E. Kuc2, A. P. Davenport2, G. A. Gray1, Y. V. Kotelevtsev1, D. J. Webb1.1Centre for Cardiovascular Science, University of Edinburgh, Edinburgh; 2Clinical Pharmacology Unit, University of Cambridge, Addenbrooke’s Hospital, Cambridge

Introduction: Inactivation of endothelin type B receptors (ETBR), though selective pharmacological antagonism or genetic mutation increases the circulating concentration of endothelin-1 (ET-1), suggesting that the ETBR plays an important role in the clearance of this peptide. By crossing floxed ETBR mice (FF/NoCre) with Tie2-Cre mice (WW/Tie2-Cre), we have generated an EC-specific ETBR KO mouse (FF/Tie2-Cre) to investigate the relative contribution of the EC-ETBR to the clearance of ET-1, without the confounding effects of receptor deletion in all other tissues.

Methods: Plasma ET-1 concentrations were measured by RIA and endothelin type A receptor (ETAR) and ETBR density was assessed using quantitative autoradiography. Clearance of ET-1 was measured by injecting a bolus of [125I]-ET-1 (0.37 pmol/mouse; 28 kBq/mouse) i.v. under anaesthesia. Arterial blood was sampled over the subsequent 2 minutes and the radioactivity in each sample was measured (cpm).

Results: Plasma ET-1 concentrations were greater in FF/Tie2-Cre samples (12.4±3.0 pg.ml−1) than in WW/-- controls (3.0±0.8 pg.ml−1) (n = 6; p<0.001). Autoradiography revealed significant downregulation of ETBR in EC-rich tissues such as lung of FF/Tie2 animals (80±21 amol.mm−2) compared to WW/-- controls (8±3 amol.mm−2) (n = 4; p<0.05), and preserved levels of ETAR expression despite higher concentrations of plasma ET-1. The AUC for the cpm-time graph was significantly increased in FF/Tie2-Cre mice [235056±21333 cpm.sec] compared to the WW/-- [86726±10968 cpm.sec] (n = 5; p<0.01). ETBR blockade of WW/-- mice resulted in an AUC unchanged from that of untreated FF/Tie2 animals (n = 4).

Conclusions: These studies indicate for the first time that the EC-ETBR is the primary receptor responsible for the clearance of ET-1.

025 POTENT AORTIC VASOCONSTRICTION MEDIATED BY CELECOXIB AND OTHER PGHS INHIBITORS IN THE ABSENCE OF NITRIC OXIDE

P. B. Anning*, B. Coles, V. B. O’Donnell.Department of Medical Biochemistry and Immunology, Wales College of Medicine, Cardiff University, Cardiff, CF14 4XN

Selective PGHS-2 inhibitors can promote thrombotic cardiovascular events through poorly-understood mechanisms. In this study we investigated the effects of PGHS inhibition on control vascular tone by murine thoracic aortic rings, and whether this is modulated by endogenous nitric oxide (NO). Immunohistochemistry indicated expression of both PGHS-1 and –2 in aortic vessel wall. Constriction and relaxation were determined using an isometric myograph and standard pharmacological techniques. Aspirin (ASP; 10μM) was without effect on endothelium-dependent (ACh) dilatation or phenylephrine (PE)-constriction, whilst celecoxib (CXB; 10 μM) had no effect on constriction, but caused a small inhibition of dilation at low dose ACh, with a small enhancement in dilation at the higher ACh concentrations. Finally indomethacin (IND; 10μM) caused a small inhibition of constriction, but had no effect on ACh-dependent dilation. These effects were minor however, and for the most part not significant. In total contrast to their small effects on tone when added alone, co-administration of IND, ASP and CXB with L-NAME caused a large significant enhancement in vasoconstriction, and further enhanced the inhibitory effects of L-NAME on ACh-dependent dilation. Finally, in the presence of PGHS inhibitors, there was a significant shift of the SNP-dependent dilation curve to the left indicating enhanced sensitivity of the contractile apparatus to exogenous NO. These data indicate that in the presence of NO, PGHS isoforms play minor roles in regulation of vascular tone in aorta. However, in the presence of L-NAME, PGHS (probably PGHS-2) promotes vascular relaxation, perhaps to compensate for the absence of NO. Importantly, inhibition of endogenous NO generation converts PGHS inhibitors agents into potent vasoconstrictors. These undesirable effects of PGHS inhibitors may contribute to the vascular side-effects of these drugs observed in patients with vascular disease, who characteristically show decreased NO bioactivity in vivo.

026 CHARACTERISATION OF VASCULAR FUNCTION IN MICE DEFICIENT IN THE COMPLEMENT REGULATOR, CD59A

V. B. O’Donnell*, B. Coles, B. P. Morgan, P. B. Anning.Department of Medical Biochemistry and Immunology, Wales College of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN

There is emerging interest in the role of inflammation in hypertension and angiotensin II-dependent signaling. The terminal complex of complement is an important contributor to the innate immune system which is found in human atherosclerotic lesions, suggesting it participates in the inflammatory response associated with vascular disease. To evaluate the potential role of complement lysis in regulation of vascular tone and angiotensin II-dependent hypertension, vascular function was characterized in mice deficient in CD59a, an important inhibitor of membrane attack by complement, that display constitutively elevated complement lysis intravascularly with a mild haemolysis (Holt et al, Blood, 2001;98:442). In vitro, aortic rings from CD59a-/- mice showed impaired vasoconstriction to phenylephrine, and significantly decreased expression of PECAM-1. However, acetylcholine- and sodium nitroprusside-dependent dilatation was identical to wild-type, and plasma nitrate/nitrite and aortic cGMP levels were unchanged. CD59-/- mice had similar basal blood pressure to wild type, and in vivo infusion with angiotensin II by osmotic minipump (1.1 mg/kg/day) caused a similar degree of hypertension in both strains over 7 days. Furthermore, there were no significant differences in angiotensin II-dependent cardiac or vascular hypertrophy between the strains. These data indicate that elevated vascular complement activity is associated with some alterations in reactivity of large vessels to vasoconstrictors and decreased PECAM-1 expression, however in vivo vascular function, including blood pressure and hypertrophic responses to a pressor dose of angiotensin II appear normal.

027 MICE WITH A KNOCKOUT FOR INDUCIBLE NITRIC OXIDE SYNTHASE ON AN OBESOGENIC DIET ARE PROTECTED FROM METABOLIC BUT NOT VASCULAR DYSFUNCTION

B. T. Noronha, S. B. Wheatcroft, J. M. Li, A. M. Shah, M. T. Kearney.Cardiovascular Division, Kings College London

Obesity an independent risk factor for the development of atherosclerosis is associated with abnormalities of insulin action and endothelial function. This study sought to explore the mechanisms underlying endothelial dysfunction in mice rendered obese using a high fat diet.

Male C57BL/6 mice and iNOS knockout mice on a C57BL/6 background were fed an obesogenic diet (35% fat, 35% carbohydrate) from weaning until 8 weeks. Body weight, epididymal fat pad mass, glucocompetence, systolic blood pressure and vasomotor responses in aortic rings ex-vivo were assessed after 4 and 8 weeks of feeding. Glucose regulation was evaluated by a Glucose Tolerance Test (1 mg/kg i.p) and an Insulin Tolerance Test (0.75 units/ kg i.p) after an overnight fast. Systolic Blood Pressure was measured by tail cuff plethysmography. Vascular responses were studied by suspending thoracic aortic rings (4 per animal) in a temperature controlled organ bath. Dose response curves were plotted for vasoconstriction to Phenylephrine before and 30 minutes after exposure to L-NMMA (a non-specific NOS inhibitor). Similarly dose response curves to acetylcholine (Ach 1 nM-10 μM) were performed before and after exposure to catalase (1250 units/ml), an enzyme that dismutes H2O2.

Results are shown in the table overleaf. Both wild type C57bl/6 and iNOS knockout mice developed comparable degrees of obesity and hypertension. Glucocompetence was however significantly better in the iNOS knockout mice than wild type mice suggesting than iNOS may play a key role in impaired insulin signalling during obesity. Vascular response to insulin (0.1 units/ml) an effect mediated by NO release was lost in wild type mice at 8 weeks but preserved in iNOS knockout mice. Basal NO production in mouse aortic rings was significantly lower in iNOS knockout mice than wild type mice fed high fat diet. Endothelial dysfunction was unmasked by catalase in both groups.

These results suggest that iNOS may play a key role in impaired insulin actions in both skeletal muscle and the vascular wall in obesity, despite being protected against impaired insulin action, mice with a knockout for iNOS had blunted acetylcholine responses compensated for by production of H2O2 in diet induced obesity.

028 TRANSCRIPTIONAL REGULATION OF THE NOX4 ISOFORM SUBUNIT OF NADPH OXIDASE IN ENDOTHELIAL CELLS

*MPitsiouni, A. Brewer, A. M. Shah, C. D. Gove.Cardiovascular Division, King’s College London, UK

A striking correlation exists between increased NADPH oxidase activity, a major source of reactive oxygen species (ROS) in cardiovascular cells, and heart disease. Several Nox isoforms, the catalytic subunit of the NADPH oxidase multi protein complex, have been identified in cardiac cells with distinct patterns of expression. Upregulation of transcription of Nox subunits is suggested to increase NADPH oxidase activity. We have cloned a human genomic fragment comprising putative Nox4 promoter upstream of a luciferase reporter and tested activity in transfected cell lines. Serial deletions of the 2 kb upstream of the human Nox4 gene revealed −300 bps to be the basal promoter in endothelial cells, HMEC-1. Similar promoter activity of the constructs in other cell types suggests that cell specific regulation of Nox4 expression is mediated by elements located elsewhere. Three areas highly homologous between human and mouse were identified within 27 kb upstream of the Nox4 gene indicating possible regulatory significance. These were cloned next to the basal human Nox4 promoter in sense (S) and antisense (AS) orientations. A 1.9 kb (−21/−19 kb) fragment reduced the basal promoter activity by 39.9±0.08% S and 51.9±0.07% AS suggesting the presence of a repressor (n⩾3, ANOVA, Bonferroni, p⩽0.0008). This is the first report of the Nox4 isoform promoter regulation further studies may inform signalling pathways for the regulation of ROS production and therapeutic manipulation.

029 A COMPARISON OF THE HYPOTENSIVE EFFECTS OF CALCITONIN-GENE RELATED PEPTIDE AND ADRENOMEDULLIN IN ANEASTHETISED MICE

N. C. Clark*, S. D. Brain.Dept. of Cardiovascular Biology & Medicine, King’s College London, SE1 1UL

CGRP and the related peptide adrenomedullin (AM) have depressor effects on blood pressure (BP), thought to be mediated through receptors composed of G-protein-linked calcitonin receptor-like (CL) receptors with receptor activity-modifying proteins 1, 2, or 3 (RAMP1, 2 or 3; McLatchie et al. 1998). Although the hypotensive effects of exogenous CGRP and AM are well characterised in many species, there have been few studies in the mouse. Here, the hypotensive effects of CGRP and AM were assessed in naturally ventilated, urethane (2.5 mg/g, i.p.) anaesthetised CD1 and RAMP2 transgenic mice. Agents were given as a bolus injection into the jugular vein, and BP was monitored via a cannula inserted into the carotid artery.

CGRP and AM produced dose-dependent hypotension but CGRP was ∼100-fold more potent than AM. In addition, whilst the responses to CGRP were reproducible with only a 15 min dose interval, those to AM required a >2 h recovery period, indicative of tachyphylaxis. To determine the receptors involved in CGRP- and AM-induced hypotension, mice were pretreated with antagonists at CL/RAMP1 and 2 receptors, BIBN4096BS and/or adrenomedullin (AM)22–52, respectively. While CGRP responses were abolished by BIBN4096BS, those evoked by AM were insensitive to both BIBN4096BS and AM22–52. In unique RAMP2 over-expressing mice CGRP-evoked hypotension was unaltered, whilst the hypotension produced by AM was significantly enhanced in RAMP2 OE mice compared with that produced in WT mice. These data demonstrate the dose-related ability of exogenous CGRP and AM to act as potent but distinct hypotensive agents in the mouse. These results suggest that CL/RAMP1 complexes are critical for CGRP-, but not AM-mediated effects, and that RAMP2 participates in AM- but not CGRP-induced hypotension.

• McLatchie et al. Nature 1998;393:333–339.

N.C. is funded by the B.H.F

030 EVIDENCE FOR THE FUNCTIONAL IMPORTANCE OF RAMP2 IN INFLUENCING ADRENOMEDULLIN VASOACTIVE RESPONSES

C. W. Tam*, Z. Lazar, W. Born, S. D. Brain.Centre for Cardiovascular Biology & Medicine, New Hunts House, Kings College London, Guy’s Campus, London SE1 1UL

The potent vasodilators adrenomedullin (AM), produced by vascular cells, and CGRP, which is structurally related and a neuropeptide, act via the CGRP receptor family. The receptors are formed from the seven transmembrane domain calcitonin receptor like receptor (CL), and one of three single transmembrane domain proteins termed receptor activity modifying proteins (RAMP). Thus, an AM receptor is formed from CL and RAMP2 whilst CL and RAMP1 form a CGRP receptor (McLatchie et al., 1998). This theory has only so far been established in cultured cell lines. Our aim is to investigate this theory in tissues isolated from RAMP2 over expressing (TG) mice. Previous studies in mouse isolated aorta have demonstrated a response to AM that was insensitive to the CGRP peptide antagonist, CGRP8–37, thus suggesting that AM acts via the CL/RAMP2 receptor in this tissue. The AM transgenic mice were generated by insertion of a SigMyc-mRAMP2 cDNA construct into fertilized oocytes and then introduced in to the genome of C57Bl6 mice. Mice negative for the transgene were used as wild type (WT) controls. Sections (∼3 mm) of thoracic aorta were isolated and mounted in a glass organ chamber and tone (>0.2 g) was induced by noradrenaline (100–300 nM). Relaxation was induced in a dose dependent manner in response to CGRP (0.3–30 nM) and AM (0.3–100 nM). The responses to CGRP were similar in both WT and RAMP2 TG mice. By comparison, AM responses were significantly (p<0.05) enhanced in terms of sensitivity and maximal responses. Additional studies using CGRP and AM antagonists provided further evidence for the CL/RAMP receptor theory. Taken together, these results support the theory that RAMP2 has functional relevance in mediating AM responses in vascular tissues.

• McLatchie LM, Lee MG, Foord S. Nature 1998;393:333–339.

C.Tam is funded by the British Heart Foundation.

031 C-REACTIVE PROTEIN IS AN INDEPENDENT PREDICTOR OF ENDOTHELIAL DYSFUNCTION IN CORONARY ARTERY DISEASE PATIENTS OPTIMALLY TREATED WITH STATINS: STUDIES IN HUMAN SAPHENOUS VEIN

A. Momin, A. Shah, D. Grieve, S. Wheatcroft, C. Driver, A. El-Gamel, J. Desai, L. John, M. Marrinan, R. Sherwood, M. Kearney.Cardiovascular Division, King’s College London

Endothelial dysfunction (ED), a key event in the initiation/progression of atherosclerosis, is common in patients with coronary artery disease (CAD) despite control of conventional risk factors. Recently, novel non-lipid risk factors for CAD have emerged, among which C-reactive protein (CRP) has been suggested to be linked to ED. However, the relationship between CRP and ED in CAD patients treated with statins who have optimal plasma cholesterol and blood pressure control is unclear. The aim of this study was to explore the role of CRP as a predictor of ED in patients optimally treated with statins. We recruited consecutive patients with CAD undergoing elective CABG surgery, excluding patients with inflammatory diseases that may lead to elevated CRP. Endothelial function was assessed ex vivo in saphenous vein (SV) rings obtained at time of CABG. Of 115 patients, 99 (mean age 66±0.9; 90% male) were optimally treated with statins, with cholesterol levels <5.0 mmol/L (mean 3.1±0.7) and triglycerides 1.2±0.1 mmol/L. These patients had BMI 26.9±0.4 kg/m2, waist circumference 99.8±1.0 cm, systolic blood pressure 138.7±2.2 mmHg, diastolic blood pressure (DBP) 74.3±1.1 mmHg, fasting glucose 6.2±0.2 mmol/L, and CRP 2.4±0.2 mg/L. Vascular reactivity of preconstricted SV rings was assessed in 77 of these patients. Maximal relaxation to the endothelium-dependent vasodilator acetylcholine (Ach; 10−9–10−4 mol/L) was 25.5±2.3% and to the endothelium-independent vasodilator sodium nitroprusside (SNP; 10−9–10−4 mol/L) was 125.8±7.0%. In univariate analysis, CRP (r = −0.3), waist circumference (r = −0.19) and BMI (r = −0.2) were significantly inversely correlated with relaxation to Ach (all P<0.05). In stepwise multivariate analysis, the only independent predictor of relaxation to Ach was CRP (r = −0.3; P = 0.025). Relaxation to SNP was not associated with any of the variables. In multivariate analysis to assess correlates of plasma CRP in this group of patients, the only independent predictors were BMI (r = 0.34), DBP (r = 0.25) and fasting glucose (r = 0.3) - all P<0.01.

Conclusion: In statin-treated CAD patients with low cholesterol levels undergoing CABG, CRP is the only independent predictor of ED. These data support a link between CRP and ED even in patients treated with current;y recommended doses of statins.

032 LEPTIN IS AN ENDOTHELIAL INDEPENDENT VASODILATOR IN HUMANS WITH CORONARY ARTERY DISEASE: EVIDENCE FOR DIVERGENCE OF RESISTANCE TO LEPTIN’S VASCULAR AND METABOLIC ACTIONS

A. Momin, A. Shah, D. Grieve, C. Driver, R. Sherwood.M Kearney Cardiovascular Division, King’s College London

Leptin has emerged as an important peptide in metabolic regulation, with obese patients often being resistant to the effects of leptin. Recently, studies have suggested that leptin may also have vasoactive effects. We explored this possibility in human patients undergoing coronary artery bypass surgery. The vasoactive effect of leptin was assessed ex-vivo in saphenous vein (SV) rings obtained at time of CABG. 131 patients undergoing CABG were recruited (age 66±0.9, (mean±SEM) 90% male).These patients had body mass index 26.8±0.4 kg/m2, waist circumference 99.2±1.0 cm, systolic blood pressure 138.6±2.0 mmHg, diastolic blood pressure 74.4±1.2 mmHg, fasting glucose 6.5±0.2 mmol/L, LDL cholesterol 1.8±.06 mmol/L, HDL cholesterol 1.0±0.2 mmol/L.Vascular reactivity to leptin (10−13–10−7M) was assessed in preconstricted SV rings in all patients (n = 131). Mean maximal relaxation to leptin was 24.5±1.6%; all rings relaxed fully to sodium nitroprusside. To explore the mechanism of leptin’s vasorelaxant effect we performed studies (n = 8) in the presence of the non-selective nitric oxide synthase inhibitor L-NMMA (10−4M). This had no effect on leptin-induced vasorelaxation (17.4±3.4 versus 17.8±3.3%; P = 0.9). Endothelial denudation also had no effect on leptin-induced vasorelaxation (17.4±4.4 versus 22.5±3%; P = 0.4). Since the central effect of leptin is thought to be mediated through potassium channels, we explored the possibility that its vascular actions are mediated in a similar fashion. In the presence of KCl 30 mmol/L to inhibit hyperpolarisation, the vasodilator effect of leptin was completely blocked (12.6±5.6 versus 0.08±4.1%; P<0.001). To assess the relationship between total body fat and leptin’s vascular effect, we assessed the correlation between body mass index (range 16.5–39.1 Kg/m2) and maximal leptin-induced vasodilatation (range 0–102.9%) in the whole population. However, there was no correlation between these variables (r = 0.05, P = 0.55). These data demonstrate for the first time that leptin is a vasodilator peptide in human veins. Moreover, the actions of leptin are endothelial- and NO-independent. These data also show that the vasoactive effects of leptin are preserved in obese patients, supporting tissue specificity of leptin resistance in humans.

033 PLAQUE RUPTURE-RELATED GENE EXPRESSION IN FAT-FED APOLIPOPROTEIN E KNOCKOUT MICE

N. Al-Shanti*, H. J. McKinnon1, C. J. Long1, D. Dunbar1, D. Mallinson1, C. L. Jackson.Bristol Heart Institute, University of Bristol, Bristol BS2 8HW; 1Organon Laboratories Ltd, Newhouse ML1 5SH

Unstable plaques are ultimately responsible for most myocardial infarctions and strokes. The molecular events that govern plaque stability are poorly defined. A better understanding of these events would aid in the identification of novel therapeutic strategies for cardiovascular disease. To this end, we utilised an apolipoprotein E knockout mouse model which is highly susceptible to spontaneous plaque rupture in the brachiocephalic artery after 8 weeks of high-fat feeding. The left common carotid artery was used as a comparator tissue, as it is resistant to plaque rupture in these mice. In order to compare gene expression in these two arteries, we used Affymetrix microarray technology to scan 12,000 genes across a range of time points (0, 5, 7, 8 and 10 weeks of high-fat feeding). This revealed 504 genes that were significantly differentially expressed. Three of these (vitamin D receptor (VDR), klotho, and CC-10) were selected for further investigation based on their expression patterns, which showed a substantial elevation at 8 weeks in the brachiocephalic artery when compared either with time zero or with the left carotid artery at 8 weeks. These data were confirmed by TaqMan PCR. Immunocytochemical staining, using antibodies validated by Western blotting, showed specific regional distributions of these proteins in unstable plaques. These data indicate that rupturing plaques show specific patterns of gene expression, and suggest novel targets for the development of plaque-stabilising therapies.

034 THE DIFFERING EFFECTS OF MATRIX METALLOPROTEINASES ON ATHEROSCLEROTIC PLAQUE INSTABILITY

J. L. Johnson*, S. J. George, A. C. Newby, G. D. Angelini, C. L. Jackson.Bristol Heart Institute, University of Bristol, Bristol, England, BS2 8HW

Matrix metalloproteinases (MMPs) are thought to be involved in the destabilization and rupture of atherosclerotic lesions. We are testing this hypothesis in apolipoprotein E (apoE) knockout mice that have been crossed with various MMP knockouts and fed a high-fat diet for 8 weeks. The present study focuses on apoE/MMP-3 (stromelysin-1), apoe/MMP-7 (matrilysin-1), apoE/MMP-9 (gelatinase B) or apoE/MMP-12 (macrophage metalloelastase) double knockouts. Plaques in the proximal 150 μm of the brachiocephalic artery were assessed for cross sectional lesion area and the frequency of healed plaque ruptures. In addition the plaque content of smooth muscle cells and macrophages were also evaluated.

These data indicate that MMP-3 and MMP-9 play a protective role, limiting plaque growth and reducing plaque rupture. Conversely, the findings suggest that MMP-12 plays a destructive role, inducing plaque growth and triggering destabilisation. Whilst MMP-7 may promote plaque instability by reducing SMC content. This challenges the concept that MMPs simply degrade matrix and thus destabilise plaques, and suggests that members of the MMP family have diverse effects on plaque stability. Therefore, development of selective MMP inhibitors is essential to enable any potential therapeutic intervention of human atherosclerotic plaque rupture and its fatal sequelae. In particular MMP-12 appears to be an attractive target.

035 DIVERGENT EFFECTS OF ASPIRIN AND CLOPIDOGREL ON PLAQUE PROGRESSION IN THE BRACHIOCEPHALIC ARTERIES OF APOLIPOPROTEIN E KNOCKOUT MICE

H. Williams*, N. Baker§, A. Poole§, C. L. Jackson.Bristol Heart Institute and §Department of Pharmacology. University of Bristol, Bristol BS2 8HW, UK

Aspirin has been suggested to reduce acute cardiovascular events, therefore we have tested it in our apolipoprotein E knockout (apoE KO) mouse model of plaque destabilisation. In order to assess the contribution of aspirin’s platelet inhibitory action to any effect on the plaque, we also tested clopidogrel. ApoE KO mice were fed high-fat diet for 8 weeks, and simultaneously received either aspirin (15 mg/kg/day, n = 65), clopidogrel (1 mg/kg/day, n = 63), or were untreated controls (n = 92). Both aspirin and clopidogrel profoundly inhibited platelet function in these mice (data not shown). Brachiocephalic artery sections were analysed by computerised morphometry. Data are shown in the table below as mean±standard error, and statistical analysis was performed by non-parametric analysis of variance.

Treatment with clopidogrel, but not aspirin, reduced plaque size, while neither treatment had any effect on plaque stability. These data suggest that inhibition of platelet function may reduce plaque progression by reduction of thrombus incorporation following plaque rupture. However as aspirin and clopidogrel inhibit platelet function similarly, we propose that aspirin may have a dual role, by also promoting plaque progression via the 5-lipoxygenase pathway, and that this blunts its beneficial actions.

036 INFLUENCE OF SCANNING FREQUENCY AND ULTRASONIC CONTRAST AGENT ON REPRODUCIBILITY OF LEFT VENTRICULAR MEASUREMENTS IN THE MOUSE

I. Sharif, T. Anderson*, D. Anderson*, D. J. Webb, G. A. Gray, W. N. McDicken*, M. A. Denvir.Centre for Cardiovascular Science and Department of Medical Physics*, University of Edinburgh, Scotland, United Kingdom

Background: The small size and fast heart rates of mice represent technical challenges to echocardiography. This study examined the influence of different scanning frequencies and ultrasonic contrast agent and on the accuracy and reproducibility of measurements of left ventricular structure and function.

Methods: Normal mouse hearts (C57BL6) were imaged under general anaesthesia at three different scanning frequencies before and after intravenous injection of the ultrasonic contrast agent (UCA), Optison®. Coronary ligation mice (CAL) and sham-operated controls (SH) were scanned at 10–22 MHz with and without ultrasonic contrast agent.

Results: Scanning frequency had no significant effect on intra- or inter-observer variation of LV measurements in normal mice under baseline conditions. Use of UCA significantly reduced estimated EF at 10–22 MHz compared to baseline (baseline 50.8±7.6% vs UCA 39.7±7.6%, p = 0.03) and significantly increased values for LV cavity dimensions (e.g. LVarea-diastole, 20.74±1.20 vs 23.23±0.98 mm2, p = 0.002). UCA significantly reduced intra- and inter-observer variation in LV ejection fraction.

Conclusions: Scanning frequency had no significant effect on reproducibility of LV measurements in the mouse but UCA significantly reduced inter-observer variation. Use of UCA could reduce the number of mice required to observe a statistically significant change in left ventricular function due to an experimental intervention.

037 PRESSURE VOLUME RELATIONS IN THE MOUSE USING CONTRAST ENHANCED ECHOCARDIOGRAPHY

I. Sharif, T. Anderson*, D. Anderson*, D. J. Webb, G. A. Gray, W. N. McDicken*, M. A. Denvir.Centre for Cardiovascular Science and Department of Medical Physics*, University of Edinburgh, UK

Background: The end systolic pressure volume relationship (Es) is acknowledged as defining systolic performance of the left ventricle independent of loading conditions. Echocardiography combined with high fidelity LV pressure recordings can be used to determine this relation. However, the reproducibility of this technique in the mouse has not previously been assessed.

Methods: Coronary ligation (CAL, n = 12) and sham operated (SH, n = 9) mice (C57BL6) were imaged under general anaesthesia at 10–22 MHz before and after intravenous injection of the ultrasonic contrast agent (UCA), Optison® during simultaneous measurement of LV pressure using a pressure-transducer tipped catheter (1.4 F, Millar Instrument Inc, USA) inserted via the right carotid artery. Intravenous sodium nitroprusside (10–40 mcg/kg/min) was used to alter loading conditions.

Results: Under baseline conditions CAL mice demonstrated a reduced Es slope (see graph) compared with SH consistent with reduced systolic function. In the presence of UCA the Es slope reduced further in CAL animals but was unchanged for SH. The inter-observer variation in Es slope was imrpoved by the use of UCA.


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Figure 1

Conclusions: Echocardiography combined with high fidelity pressure recordings in the mouse permits the assessment of the pressure volume relation in the mouse, reproducibility of this measurement was significantly improved by the use of UCA.

038 CONDUCTANCE CATHETER FOR IN-VIVO CARDIAC FUNCTION IN MICE - IS IT SENSITIVE ENOUGH?

K. J. Dighe*, G. S. Kanaganayagam, M. S. Marber.Cardiovascular Division, King’s College London, SE1 7EH

We questioned whether the conductance catheter is a sensitive method to determine in-vivo cardiac function in mice. We conducted pressure volume analysis (Millar catheter, SPR-839) on mice with varying degrees of cardiac dysfunction at 4 or 10 weeks of remodelling (R) following permanent LAD occlusion (∼40%MI) at steady state and at varying preload. See table; Shams  = S.

Conclusion: Although we obtained significant remodelling using parameters recorded at steady state, those recorded under varying preload (such as IVC occlusion until ESP dropped to ∼60 mmHg) seemed less sensitive and specific. It is unclear at present whether this is a result of technique or data acquisition and processing.

039 IS THERE OBSTRUCTION OF THE AORTIC VALVE USING A CONDUCTANCE CATHETER IN A MOUSE MODEL?

K. J. Dighe, M. E. Faircloth, J. E. Clark*, G. S. Kanaganayagam, M. S. Marber.Cardiovascular Division, Kings College London, Rayne Institute, St. Thomas’ Hospital, London. SE1 7EH

Pressure volume loops generated using an ultra-miniature conductance catheter are increasingly used to measure cardiac contractility in murine models. Catheters are introduced in the left ventricle (LV) either by LV puncture or following carotid (ICA) cannulation by crossing the aortic valve (AV). We questioned whether the catheter (Millar Instruments, Texas, USA), although small might cause obstruction when placed across the murine AV since the catheter occupies approximately 45% of the cross-sectional area of the aortic valve. Using the ICA route we simultaneously measured aortic pressure (AoP) using left ICA cannulation and LV pressure (LVP) using either direct LV puncture (DLVP, n = 6) or using right ICA cannulation (ICAC, n = 6). The results are described in the figure.


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Figure 2

No statistically significant differences were seen between the groups. Furthermore, the trend towards an increased AV gradient in the crossed group is of no functional significance. After this preliminary study with relatively small sample sizes we conclude that this technique does not compromise cardiac function or physiology despite the catheter occupying a significant part of the aortic orifice. This finding ratifies the use of these catheters in murine cardiovascular models. Since these models form an important and increasingly utilised tool in the study of cardiac disease, more credence may be given to results obtained by this technique.

040 SUSCEPTIBILITY OF MITOCHONDRIA TO PALMITATE IS INCREASED IN THE AGED HEART

J. Sample*, J. G. F. Cleland, A-ML. Seymour.Depts of Biological Science and Cardiology, University of Hull, HU6 7RX

Age is a major risk factor for heart failure. However, the underlying cellular mechanisms responsible for this increased susceptibility are yet to be defined. Mitochondria play a central role in cardiac metabolism and apoptotic cell death and dysregulation of both of these processes has been shown to be implicated in the transition to failure. Mitochondrial dysfunction in the senescent heart may contribute to impaired cardiac function and an increased susceptibility to injury. The aim of this study was to investigate the impact of palmitate on the mitochondrial membrane potential (Δψm) in isolated cardiomyocytes. Ventricular myocytes were isolated by collagenase digestion from 6 (n = 4) and 24 (n = 6) month old male Wistar rats. Cells were incubated with 500 µM palmitate (37°C, 100% O2) for 4 hrs and stained with 0.5 µM JC-1 or 0.5 µM Rhodamine 123. Δψm was studied using confocal microscopy. Exposure of aged cardiomyocytes to palmitate for 4 hrs resulted in a marked reduction in the JC-1 red fluorescence (figure) and an increase in Rhodamine 123 fluorescence, which indicates a decline in the number of energised mitochondria. In contrast, no significant change in fluorescence was observed in 6 month cells. These results suggest that palmitate can adversely affect mitochondrial function in the ageing heart and that this may be an important contributory factor to the development of heart failure.


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Figure 3

041 ENDOTHELIAL PROGENITOR CELLS ARE FOUND IN RESOLVING VENOUS THROMBUS

B. Modarai*, B. Sawyer, K. G. Burnand, A. Smith.Academic Department of Surgery, King’s College, London SE1 7EH

This study aimed to determine whether recanalisation during venous thrombus resolution depends on angiogenesis or de novo neovascularisation from circulating endothelial progenitors.

Irradiated mice were reconstituted with bone marrow from transgenic donors expressing green fluorescent protein (GFP) linked to the Tie2 promoter. Thrombi were formed in 2 groups of 6 mice. GFP expressing cells were located in sections of thrombi taken after 7 and 14 days. Fluorescent cell numbers were quantified and GFP expression co-localised with the macrophage marker, MAC3. FACS analysis, using the anti-endothelial antibodies, CD34 and VEGFR2, was also carried out on blood from 3 cohorts of wild-type animals that had either a thrombus induced (n = 18), a sham operation (n = 18), or no operation (n = 10).

Sections of thrombus from all transplanted animals contained GFP expressing cells around the perimeter of the thrombus at day 7. By day 14 the cells were found throughout the thrombus and their number had increased by 3-fold (p = 0.0022). No GFP expressing cells were found lining the new vascular channels that formed at either time interval. Approximately two thirds of the GFP expressing cells also expressed MAC3. Almost twice as many circulating CD34+/VEGFR2+ cells were found at day 3 in animals with thrombus compared with sham controls (p = 0.0459).

Bone marrow-derived, Tie2 expressing, cells are recruited into the thrombus during resolution. These cells did not appear, however, to line the new channels in the thrombus. The cells also expressed a macrophage phenotype and may represent a population of plastic stem cells that orchestrate thrombus recanalisation.

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Abstract 034. Effect of MMP knockout on plaque characteristics (n = 24 per group and all differences p<0.05)

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