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The BSCR is sponsored by

The Bulletinof

The British Society for Cardiovascular ResearchRegistered Charity Number: 1011141

Vol. 20 No. 2 April 2007

www.bscr.org

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The BulletinThe Publication of The British Society for Cardiovascular Research

EditorsDr Helen Maddock

Physiological and Clinical InterventionsFaculty of Health and Life Sciences

James Starley Building, Coventry UniversityPriory Street

Coventry CV1 5BFTel: 024 76 888163 Fax: 024 76 888702

E-mail: h.maddock@coventry.ac.uk

Dr Nicola SmartMolecular Medicine UnitInstitute of Child Health

30 Guilford StreetLondon WC1N 1EH

Tel.: 020 7905 2242 Fax: 020 7404 6191 E-mail: N.Smart@ich.ucl.ac.uk

Chairman

Professor David EisnerUnit of Cardiac Physiology, University of Manchester

3.18 Core Technology Facility46 Grafton Street

Manchester M13 9NTTel.: 0161 275 2702 Fax: 0161 275 2703

E-mail: eisner@man.ac.uk

Secretary

Professor Barbara McDermottDepartment of Therapeutics and Pharmacology

The Queen's University of BelfastWhitla Medical Builiding

97 Lisburn RoadBelfast BT9 7BL

Tel.: 028 90 272242/335770 Fax: 028 9043 8346E-mail: b.mcdermott@qub.ac.uk

Treasurer

Dr Michael J. CurtisCardiovascular Research

Rayne Institute, St. Thomas' HospitalLondon SE1 7EH

Tel.: 020 7188 1095 Fax: 020 7188 3902E-mail: michael.curtis@kcl.ac.uk

BAS Representative

Dr Chris NewmanClinical Sciences CentreUniversity of Sheffield

Northern General HospitalHerries Road

Sheffield S5 7AUTel: 0114 271 4456 Fax: 0114 261 9587

E-mail: c.newman@sheffield.ac.uk

CommitteeDr Andrew Baker

BHF Glasgow Cardiovascular Research Centre Division of Cardiovascular and Medical Sciences

University of Glasgow, Western Infirmary Glasgow G11 6NT

Tel: 0141 211 2100/2116 Fax: 0141 211 1763 E-mail: ab11f@clinmed.gla.ac.uk

Dr Katrina BicknellSchool of Pharmacy, The University of Reading

PO Box 228, Whiteknights Reading, Berkshire RG6 6AJ

United Kingdom Tel: 0118 378 7032 Fax: 0118 931 0180

E-mail: k.bicknell@rdg.ac.uk

Dr Barbara Casadei University Department of Cardiovascular Medicine

John Radcliffe Hospital, Oxford OX3 9DU

Tel: 01865 220132 Fax: 01865 768844E-mail: barbara.casadei@cardiov.ox.ac.uk

Dr Andrew Grace Section of Cardiovascular Biology

Department of Biochemistry, University of Cambridge Tennis Court Road

Cambridge CB2 1QW Tel: 01223 333631 Fax: 01223 333345

E-mail: ag@mole.bio.cam.ac.uk

Dr Gillian A. GrayEndothelial Cell Biology and Molecular Cardiology Group

Centre for Cardiovascular ScienceQueen’s Medical Research Institute, University of Edinburgh

47 Little France Crescent,Edinburgh EH16 4TJTel: 0131 242 9213

E-mail: gillian.gray@ed.ac.uk

Dr Cathy HoltDivision of Cardiovascular and Endocrine Sciences

University of Manchester3.31b Core Technology Facility

46 Grafton Street, Manchester M13 9NTTel: 0161 275 5671 Fax: 0161 275 1183

E-mail: cathy.holt@manchester.ac.uk

Dr Chris JacksonBristol Heart Institute University of Bristol

Level 7, Bristol Royal Infirmary Bristol BS2 8HW.

Tel/Fax: 0117 928 2534 E-mail: chris.jackson@bristol.ac.uk

Dr Nicola King Institute of Medicine

Universiti Brunei Darussalam Jalan Tungku Link, Gadong BE 1410

Brunei Darussalam.Tel: 00673 8989370

Email: nautilus_500@hotmail.com

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Editorial 3 Review: 'Structural analysis of cardiac muscle by modern electron microscopy'

by Dr Pradeep K. Luther 4

Viewpoint: 'The prospects for gene therapy in the management of peripheral vascular disease' by Alex Rossdeutsch 11

BSCR Autumn 2007 Meeting: Programme 16

Cardiovascular Related Meetings 17

British Heart Foundation Grants 18

Cardiovascular Related Wellcome Trust Grants 19

BSCR Spring 2007 Meeting: 'The QT Interval and drug-induced torsades de pointes' 20

Editorial

Helen Maddock and Nicola Smart

Cover artwork copyright Anthony Wright, �997Cover design copyright Siân Rees and Anthony Wright, �997

Contents

Welcome to the April 2007 issue of The Bulletin!

Our review article for this issue, written by Dr Pradeep Luther of Imperial College London, provides an informative overview of the applica-tion of electron microscopy to the study of cardiac muscle. Dr Luther draws upon examples from his own research work to illustrate the enormous value of the technique as exemplified in some of his ex-quisite electron micrographs.

Alex Rossdeutsch, a MBPhD student at University College London, offers his views on 'The prospects for gene therapy in the management of peripheral vascular disease'. In an insightful and thought-provoking essay, Alex discusses the hopes and current limitations of this technology and his

opinions on the way forward for gene therapy. We are sure you'll agree that this type of article makes for fascinating reading and we also know that BSCR members like to make their opinions known. If you feel inspired to write a similar article and to share your views with Bulletin readers, please contact us. We'd be delighted to publish your work.

We are pleased to be able to announce details of the Autumn BSCR Meeting, organised by the Treasurer, Dr Michael Curtis. Mike has put together a stimulating set of symposia on drug-induced tor-sades de pointes. The full programme is included within and further practical details can be found on the back cover and on the BSCR website.

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Structural analysis of cardiac muscle by modern electron microscopy

by Dr Pradeep K Luther, Biological Nanosciences Section, National Heart & Lung Institute, Imperial College London,

Revival of electron microscopyBy the end of the 80s, the popularity of electron

microscopy (EM) as a tool for structural analysis in cell biology was very much in the wane, resulting in EM units being shut down in many educational and commercial organisations (Griffiths, 2001). This reversed in the 90s and the trend continues unabated. The revival of electron microscopy is due to the devel-opments in the technology for the electron microscope. These include very bright and coherent electron sources (field emission gun), cryoholders to allow imaging of frozen samples with improved resistance to radiation damage and high resolution CCD cameras for recording images directly to computer. These new microscopes are also highly automated. These innovations have been brought about by the needs of new techniques that have emerged in the last two de-cades, including cryo-electron microscopy for imaging samples such as macromolecular assemblies rapidly frozen in the native state. Another modern technique is the so-called "single particle analysis", in which images of thousands of "particles" (macro-molecular assemblies), are classified for their 3D orientation, averaged and combined to give 3D images (van Heel et al., 2000). Spectacular successes have been achieved giving high resolution, better than 1 nm, 3D images, allowing fitting of atomic structures of components in the 3D maps (Orlova and Saibil, 2004).

Electron tomography is another new technique served well by the new microscopes, providing 3D images of samples where only 2D information was available before (Koster et al., 1997; Lucic et al., 2005; McIntosh et al., 2005). The resolution in the depth of the sample, ~5-10 nm, is a marked improvement over the simple 2D image of a section 50-100 nm thick. The automated operation of the new microscopes allows efficient collection of tilt series: images of a sample tilted by small steps over the full tilt range available (~ ±70o). Electron tomography is especially applicable to multi-cellular samples like the heart, that have to be sectioned thin enough for imaging in the electron microscope.

Benefit of electron microscopy for cardiac research

The reader may wonder whether electron mi-croscopy can really benefit our basic understanding of cardiac function. That this is the case is illustrated by one of the projects in my lab. Myosin binding protein C (MyBP-C) is a 130 kD protein that is located along at least seven stripes of separation 43 nm in each half A-band (Fig 1) (Squire et al., 2005). Mutations in MyBP-C are a major cause of familial hypertrophic cardiomyopathy (Richard et al., 2003). To understand how MyBP-C can have such a profound effect on heart function, we first need to understand the arrangement of the protein in normal cardiac muscle and then compare with diseased tissue or a mouse model of the disease (Harris et al., 2002; Yang et al., 1998). This is being studied in our laboratory by a British Heart Foundation funded project using electron tomography. Described later in this review is the application of elec-tron tomography to understand the 3D structure of the I-band in cardiac muscle. Thus electron microscopy in building up the detailed structure of the cardiac sarcomere will help to elucidate the interactions that occur in the activity of the cardiomyocyte.

Electron microscopy of rat cardiac muscleIn this section EM methods for rat heart are

described and they are applicable to other small mammals, especially the mouse, as it is the focus of much transgenic work. However mouse heart is more delicate than rat heart and quality electron microscope images are harder to achieve. Our preferred muscles are the papillary and trabaculae muscles of the right ventricle as they are thin V-shaped or narrow colum-nar. For dissection of the heart, the medium is Krebs solution (comprising in mM: NaCl, 94.5; KCl, 5; NaHCO3, 25; Na2HPO4, 1; MgSO4, 1; NaAcetate, 10; Glucose, 11; CaCl2, 1) with 30mM 2,3-butane-dione monoxime (BDM). BDM inhibits muscle contraction and has a protective effect on the muscle during dissection (Kiriazis and Gibbs, 1995; Yagi et al.,

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1992). The right ventricular wall is opened to expose the papillary muscles. Fine silk is tied to the papillary muscle and the muscle excised from the heart. The isolated muscle is then tied to micro-pins on a petridish filled with Sylgard 84 gel. The medium is replaced with Krebs and the oxygenation continued. Then the muscle is fixed with 3% glutaraldehyde in Krebs for 1 hour. After rinsing, the muscles are processed for EM by two methods: conventional and cryosectioning.

The conventional method involves fixation in 1% osmium tetroxide, dehydration in acetone series and embedding in Araldite. Fig 2a shows an example of an electron micrograph of a thin ~100 nm section of rat papillary muscle, stained with uranyl acetate and lead citrate. All the components in the micrograph can be identified by comparing with the schematic in Fig 1.

For cryosectioning using the Tokuyasu method (Griffiths, 1993; Luther and Morris, 2003), the muscle is incubated in 2.3M sucrose solution in Krebs for 2-3 days. Small, ~1mm3, pieces are cut and mounted on "cryo-pins" and frozen by plunging the pin into liquid nitrogen. Sections are cut using a cryo-ultramicrotome such as the RMC MT7 with a CR20 cryochamber or a Leica EM UC6 ultramicrotome fitted with an EM FC6 cryochamber. The cryosections are transferred to a carbon-coated EM grid, thawed and floated on a

drop of buffer. At this stage, we treat the grid in one of two ways. A novel method favoured by our lab is to treat the floating grid as a sample for cryo-electron microscopy (see later; Luther and Morris, 2003). For routine EM, the grid is negative stained with uranyl acetate or ammonium molybdate, dried and viewed in the electron microscope at room temperature. An example of such a section is shown in Fig 2b.

Assessment of order in striated muscleThe beautifully patterned nature of striated

muscle gives us objective methods to assess the order and preservation of the native structure. On a quali-tative basis, we look for strong well-defined straight M-bands and Z-bands with clear stripes and sharp well-defined edges of the A-bands. Across the sec-tion, the adjacent myofibrils should be aligned with matching Z-bands (Z-lines) and M-bands. The order in the micrograph can be assessed quantitatively by calculating the Fourier transform of a digitised A-band as shown in Fig 2c for a plastic section or Fig 2d for a cryosection. The Fourier transform can be calcu-lated with image processing software, e.g., ImageJ (Rasband, 1997). These transforms can be compared with the X-ray diffraction pattern of a live muscle as shown in Fig 2e. In these patterns, the fibre axis is up the page. The meridional spot corresponding to

Figure 1: Components of a vertebrate muscle sarcomere. The length of the A-band is about 1.6µm.

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14.3 nm (3rd order of the 42.9 nm repeat of myosin crossbridges) is marked with an arrow in each figure. Each of the processing steps for electron microscopy reduces the order in the sample; clearly the cryosec-tion (Fig 2d) shows much higher order detail than the plastic section (Fig 2c). The X-ray pattern in Fig 2e, provided by Dr. Pieter de Tombe, has exceptionally high quality for cardiac muscle, although such qual-ity is well-established for skeletal muscle (Squire et al., 2006). Improved preservation in skeletal muscle for electron microscopy studies has been obtained by fast freezing a fibre by propelling it towards a liquid helium-cooled copper block followed by freeze-sub-stitution (Padron et al., 1988).

Electron microscopy of human cardiac muscle Unlike the fibre-oriented muscles like the pap-

illary in small animals, the samples of human tissue are small pieces from the heart wall. For histology or electron microscopy of cardiac muscle, it is important to orient the heart sample so that sections are cut along (or perpendicular to, if necessary) the myofibrils. Care has to be taken in this step as muscle fibres have various orientations in the heart wall. Cardiac tissue deteriorates rapidly and addition of BDM in the initial stages of muscle preparation has a protective effect on the muscle.

Human cardiac muscle is precious; the samples in our work were obtained from myectomy operations or from one explant heart. The miniscule

Figure 2: Electron microscopy of rat car-diac muscle (right ventricle papillary). (a) Plastic section, ~100 nm thick, of convention-ally processed sample (glutaraldehyde/os-mium fixation, dehydration, embedding in Araraldite), positive stain with uranyl acetate and lead citrate. (b) Cryosection, negative stain with uranyl acetate (image contrast re-versed for this figure). The prominent stripes running across the A-band are due to myosin crossbridges and myosin binding protein C (MyBP-C). Much greater detail is preserved in the cryosection compared with the plastic section. (c,d,e) Comparison of the X-ray dif-fraction pattern (e) with the Fourier transform (c) of the plastic section A-band and the Fourier transform (d) of the cryosection. The fibre orientation is up the page. In each figure, the arrows refer to the spot corresponding to the crossbridge spacing of 14.3 nm. The extent of the spots in the Fourier transforms is a direct measure of the resolution in the electron micrographs; hence the cryosection clearly has much higher resolution. Scale bar for (a) and (b) = 0.5µm. X-ray diffraction pat-tern of rat papillary muscle kindly provided by Dr. Pieter de Tombe.

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amounts given to selected researchers are fortunately sufficient for electron microscopical analyses once the technique is established. Fresh unfrozen human cardiac tissue is indeed very difficult to procure. My cardiologist collaborator obtained samples at myec-tomy operations and immediately put the piece for EM into Krebs/30 mM BDM solution and freezing the rest into liquid nitrogen. Back in the EM laboratory, we fixed a small piece in 3% glutaraldehyde in Krebs, for ~1 hour. After rinsing out the fixative, the cardiac piece

Figure 3. Human cardiac fibres from a failing heart (dilated cardiomyopathy, explant heart). The frozen sample was thawed in relaxing solu-tion, homogenised, applied to a cover-slip with a Cytospin and then conventionally processed for EM (see text for details). (a) Light micrograph showing the general morphology of homogenised fragments. (b) Electron micrograph showing good order in the A-band. The quality of the electron micrographs of human samples is very variable, and frequently samples comprise very short, ~1.4 µm, sarcomere lengths and disrupted Z-lines. Scale bar for (b) = 0.5µm

Figure 4. Cryo-em of refrozen cryosections. For details see text. Reproduced with permission from (Luther and Morris, 2003).

was examined under a well-lit dissecting microscope to identify flat surfaces, indicative of fibre orientations. It was sliced parallel to these surfaces and then processed for conventional plastic sections or cryosections.

Human tissue is mostly frozen immediately into dry ice or into liquid nitrogen. Although the freez-ing is likely to rupture the membranes of many cells, we can surprisingly still get good structural detail from such samples. This may be due to the cryoprotective effect of the cytoplasm within the myocytes. For EM processing of the frozen samples, a piece is first thawed by immersing it into a solution of ice-cold relaxing solution (strength in mM: KCl, 100; Imidazole, 20; MgCl2, 7; EGTA, 2; ATP, 4; pH=7.0) (Patel et al., 2001). It is then homogenised for 4 seconds using a Polytron homogeniser. The fragmented myocytes are examined in a light microscope with a x40 objective to ensure that the fragments are thin enough to show clear striations. The suspension is centrifuged, immersed in skinning solution (relaxing solution + 1% Triton),

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Figure 5. Electron tomography of striated muscle. (a,b). Rat papillary muscle. (a) Transverse section electron micrograph. Two serial sections were used. (b) Surface rendered view showing actin filaments. (c,d,e) Fish skeletal muscle in rigor. (c) Longitudinal section electron micrograph. (d) One 2D slice from the 3D image (e) Model made with IMOD showing the main features of the tomogram. Figure kindly provided by Thomas Burgoyne.

and then subjected to two cycles of centrifugation and rinses in relaxing solution (Patel et al., 2001). It is then applied to a plastic cover-slip with a Cytospin (Shandon) and then conventionally processed for EM. Fig 3(a) shows a light micrograph illustrating the morphology of homogenised myocytes. Fig 3(b) shows an electron micrograph showing good order in the A-band. The quality of the electron micrographs of human samples is very variable, and frequently samples show very short, ~1.4 µm, sarcomere lengths and disrupted Z-lines. In problem cases, incubation of the sample with a BDM solution gives patches of reasonable length sarcomeres (~ 2 µm).

Cryoelectron microscopy of refrozen cryosec-tions

Cryoelectron microscopy (cryo-em) of frozen macromolecular assemblies has provided excellent 3D images at high <1nm resolution (Orlova and Saibil, 2004). To apply cryo-em to a multicellular sample like the heart, the sample has to be frozen, sectioned with

a cryo-ultramicrotome and the sections viewed frozen in the electron microscope. Although great progress has been made in this field (Al-Amoudi et al., 2004), it remains a demanding technique. A major problem with the method is that the process of sectioning pro-duces mechanical artefacts like creases, knife marks, fractures and compression. In a recent study, we described an alternative method (Luther and Morris, 2003). Simple cryosections of chemically fixed rat papillary muscle were cut, applied to a carbon coated grid, thawed and floated on a drop of buffer. The grid was then used to produce a cryo-sample: it was rapidly frozen and examined in an FEI 200 KV field emission gun electron microscope. The results are shown in Fig 4. The low magnification overview in Fig 4a shows good contrast for the unstained sample, and shows all the main features of striated muscle: sarcomeres (S), A and I bands (A,I), Z-bands (Z) and M-bands (M). A row of mitochondria (Mc) is outlined. A zoomed-in view of a single sarcomere is shown in Fig 4b. This shows excellent detail in the M-band (M) and A-band. Individual myosin filaments (MF) are pointed out.

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What is particulary impressive in these cryo-em images is that the mechanical artefacts seen in cryo-em im-ages of frozen (unthawed) sections are absent in these refrozen cryosections. It is likely that the process of thawing relaxes the section and removes the locked-in defects. The Fourier transform of the boxed region in (b) is shown in the inset (c). The spots corresponding to the 3rd and the 9th order of the 43 nm repeat are pointed with small and large arrows respectively. We have used a refrozen cryosection to obtain a complete tilt series and calculated the tomogram (data not shown here). Hence we believe the method of refrozen cryo-sections holds great potential to reveal detailed insight into 3D structure.

Electron tomography of the I-band Electron tomography refers to the process of

producing 3D images of a sample from a set of images obtained by tilting the sample in the electron micro-scope by small increments over the full tilt range, ~ ± 70o (tilt series). It has the potential to improve depth resolution of a sample to 5-10 nm compared with the simple 2D image of ~100 nm thick sample. In this section, we describe the application of tomography to study the cardiac muscle I-band, a current PhD project in our laboratory. Fig 5a shows an electron micrograph of a transverse section, ~100 nm thick, of rat papil-lary muscle. A second transverse section, serial to this region was also used. For both, tilt series images were obtained about two perpendicular tilt axes. Such dual axes tilt series cover the maximum 3D volume of a sample. The tomograms were calculated for the two serial sections and combined using the software IMOD (Mastronarde, 1997). Fig 5b shows a surface-rendered side-view of the combined tomogram. It shows irregular looking thin filaments. The noisy nature of tomograms is typical. To help in interpreting the tomography images, we apply image denoising and averaging techniques (Lucic et al., 2005).

In Fig 5c-e, longitudinal section tomography of the I-band is described. Here the sample is fish skeletal muscle, selected for the short I-band and rigor stretched thin filaments. Fig 5d shows a thin 2D slice from the tomogram, showing a central Z-band, with fine thin filaments emanating to the left and right and overlapping with the denser myosin filaments. Quite regular crossbridges can be seen linking the actin and myosin filaments. To understand the tomogram, the outline of the main features can be traced (painstak-ingly!) in different colours through each of the 2D slices of the 3D image stack. This is shown in Fig

5e. Such tomograms are being used to understand the path of the thin filament through the I-band, to find out the rules for the transformation of the square lattice at the Z-band to the hexagonal lattice in the A-band and to discover what other components are present in the I-band.

Concluding Remarks To see a cardiomyocyte under the light micro-

scope with its zebra stripes and then to see it contract-ing spontaneously, is to fall in love with this cell type forever. The sarcomere is the repeating unit of the cardiomyocyte. We have shown in this review how electron microscopy is helping to elucidate the fine structure of the cardiac muscle sarcomere.

Acknowledgements I am grateful to Cathy Timson for expert EM

work, to Thomas Burgoyne for providing the tomog-raphy images for Figure 5 and to Daniel Moore for expert workshop help (preparing cryo-pins etc). I am indebted to several scientists for providing cardiac samples and lots of advice: Jon Kentish, Sonya Bard-swell, Todd Herron, Jitendra Patel, Sian Harding, Peter O'Gara, Adam Jacques (my cardiologist collaborator), Steve Marston and John Squire.ReferencesAl-Amoudi, A., J.J. Chang, A. Leforestier, A. McDowall, L.M. Salamin, L.P. Norlen, K. Richter, N.S. Blanc, D. Studer, and J. Dubochet. 2004. Cryo-electron microscopy of vitreous sections. Embo J. 23:3583-8.

Griffiths, G. 1993. Fine structure immunocytochemistry. Springer-Verlag, Berlin.

Griffiths, G. 2001. Bringing electron microscopy back into focus for cell biology. Trends Cell Biol. 11:153-4.

Harris, S.P., C.R. Bartley, T.A. Hacker, K.S. McDonald, P.S. Douglas, M.L. Greaser, P.A. Powers, and R.L. Moss. 2002. Hypertrophic cardiomyopathy in cardiac myosin binding protein-C knockout mice. Circ Res. 90:594-601.

Kiriazis, H., and C.L. Gibbs. 1995. Papillary muscles split in the presence of 2,3-butanedione monoxime have normal energetic and mechanical properties. Am J Physiol. 269:H1685-94.

Koster, A.J., R. Grimm, D. Typke, R. Hegerl, A. Stoschek, J. Walz, and W. Baumeister. 1997. Perspectives of molecular and cellular electron tomography. J Struct Biol. 120:276-308.

Lucic, V., F. Forster, and W. Baumeister. 2005. Structural stud-ies by electron tomography: from cells to molecules. Annu Rev Biochem. 74:833-65.

Luther, P.K., and E.P. Morris. 2003. Cryoelectron microscopy of refrozen cryosections. J Struct Biol. 142:233-40.

Mastronarde, D.N. 1997. Dual-axis tomography: an approach

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with alignment methods that preserve resolution. J Struct Biol. 120:343-52.

McIntosh, R., D. Nicastro, and D. Mastronarde. 2005. New views of cells in 3D: an introduction to electron tomography. Trends Cell Biol. 15:43-51.

Orlova, E.V., and H.R. Saibil. 2004. Structure determination of macromolecular assemblies by single-particle analysis of cryo-electron micrographs. Curr Opin Struct Biol. 14:584-90.

Padron, R., L. Alamo, R. Craig, and C. Caputo. 1988. A method for quick-freezing live muscles at known instants during contraction with simultaneous recording of mechanical tension. J Microsc. 151 ( Pt 2):81-102.

Patel, J.R., D.P. Fitzsimons, S.H. Buck, M. Muthuchamy, D.F. Wieczorek, and R.L. Moss. 2001. PKA accelerates rate of force development in murine skinned myocardium expressing alpha- or beta-tropomyosin. Am J Physiol Heart Circ Physiol. 280:H2732-9.

Rasband, W.S. 1997. ImageJ. U.S. National Institutues of Health, Bethesda.

Richard, P., P. Charron, L. Carrier, C. Ledeuil, T. Cheav, C. Pichereau, A. Benaiche, R. Isnard, O. Dubourg, M. Burban, J.P. Gueffet, A. Millaire, M. Desnos, K. Schwartz, B. Hainque, and M. Komajda. 2003. Hypertrophic cardiomyopathy: distribution of disease genes, spectrum of mutations, and implications for a molecular diagnosis strategy. Circulation. 107:2227-32.

Squire, J.M., H.A. Al-Khayat, C. Knupp, and P.K. Luther. 2005. Molecular architecture in muscle contractile assemblies. Adv Protein Chem. 71:17-87.

Articles for The Bulletin

Would you like to write a Review or Laboratory Profile for the BSCR Bulletin? These articles provide an excellent opportunity to let BSCR

members know about your research activities and also provide an insight into

your research field.

We are keen to hear from anyone in Cardiovascular research who would be willing to write for The Bulletin. If you

are interested, please contact the Bulletin editors with your ideas:

Helen (h.maddock@coventry.ac.uk) or Nicola (N.Smart@ich.ucl.ac.uk)

Volume Date Deadline

�0 (�) July2007 ��th May

�0 (�) October2007 �st September

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Submission Deadlinesfor

The Bulletin:

Dr Pradeep K. Luther is a Principal Research Fellow at the Biological Structure & Function

Section, Biomedical Sciences Division Sir Alexander Fleming Building

Imperial College London Exhibition Road

London SW7 2AZ E: p.luther@imperial.ac.uk

W: www.sarcomere.org

Squire, J.M., T. Bekyarova, G. Farman, D. Gore, G. Rajkumar, C. Knupp, C. Lucaveche, M.C. Reedy, M.K. Reedy, and T.C. Irving. 2006. The myosin filament superlattice in the flight muscles of flies: A-band lattice optimisation for stretch-acti-vation? J Mol Biol. 361:823-38.

van Heel, M., B. Gowen, R. Matadeen, E.V. Orlova, R. Finn, T. Pape, D. Cohen, H. Stark, R. Schmidt, M. Schatz, and A. Patwardhan. 2000. Single-particle electron cryo-microscopy: towards atomic resolution. Q Rev Biophys. 33:307-69.

Yagi, N., S. Takemori, M. Watanabe, K. Horiuti, and Y. Amemiya. 1992. Effects of 2,3-butanedione monoxime on contraction of frog skeletal muscles: an X-ray diffraction study. J Muscle Res Cell Motil. 13:153-60.

Yang, Q., A. Sanbe, H. Osinska, T.E. Hewett, R. Klevitsky, and J. Robbins. 1998. A mouse model of myosin binding protein C human familial hypertrophic cardiomyopathy. J Clin Invest. 102:1292-300.

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In recent years, peripheral arterial disease (PAD) has been held up as a promising example of a disease which might be amenable to gene therapy strategies. The term "peripheral arterial disease" commonly refers to occlusive ischaemic pathology of arteries in the lower limb. PAD is further subdivided according to the severity of symptoms and clinical findings. "Intermittent claudication" (IC) describes the symptoms of ischaemic pain in the legs on exercise which disappear at rest and is usually accompanied by an ankle:brachial blood pressure ratio (ABPI) of <0.9. Its occurrence in the Western world is usually the result of atherosclerotic change in the sub-aortic arteries of the lower limb. IC may progress to "critical limb ischaemia" (CLI) if occlusion becomes worse so that ischaemic pain is felt at rest and is often associated with an ABPI <0.6. As well as being significant causes of morbidity, IC and CLI patients are at an increased risk of developing acute limb ischaemia (ALI) as a result of sudden thrombosis or thromboembolism of previously diseased vessels1. Current best practice management of PAD differs depending on the stage of disease and is summarised in Fig.1. Currently, no therapy offers a "cure" for the disease2 and so in recent years there has been much focus on novel strategies to alleviate the symptoms and pathology of PAD. Observations that patients first presenting with IC often undergo a temporary improvement in symptoms due to collateral vessel formation around the point of occlusion has led to attention being lavished upon the possibility of therapeutically inducing new vessel formation to by-pass damaged arteries3. It has been postulated that the exploitation of angiogenesis might be one means of achieving this aim. Angiogenesis is the process by which new vasculature can sprout from an already established vessel. This process is controlled by a multitude of growth factors, some of whose endogenous functions are poorly understood.

Gene therapy in its simplest terms involves the vector-mediated transfer of genetic material to the so-

matic cells of an individual in order to allow expression of a therapeutic molecule(s) by those same somatic cells4. Gene therapy in PAD has centered on the trans-fection of genes encoding angiogenic growth factors into the diseased vasculature. There are many potential benefits of using gene therapy over the administration of recombinant protein which have been used to justify investigations into the use of gene therapy in this area. First of all, if fully optimised, continued gene expres-sion within a cell could in theory ensure an unbroken supply of growth factor at a concentration constantly within the therapeutic window. Additionally, gene therapy might overcome the technical and economi-cal challenges of mass-producing large quantities of highly purified human recombinant protein5.

Proof of concept experiments have been con-ducted largely in animal models of limb ischaemia, primarily using genes expressing isoforms of vascular endothelial growth factor (VEGF). VEGF plays a variety of non-redundant roles in the process of angio-genesis. A VEGF mediated increase in vascular per-meability is the initial trigger for the extravasation of plasma proteins to allow the construction of a scaffold for migrating endothelial cells6. The different subtypes of VEGF then provide a stimulus for the migration and proliferation of endothelial cells onto this scaffold7. One study of note which precipitated the first trials in humans involved excision of the femoral artery in rabbits to create a model of unilateral hind limb ischaemia. Plasmid DNA encoding the VEGF165 isoform was then used to coat the hydrogel polymer of an angioplasty balloon and was delivered percutane-ously to the femoral artery of the rabbit. Ultimately, it was observed that such treatment improved limb perfusion as manifested by a significant improvement in the ratio of calf blood pressures between ischaemic and healthy limbs, vasodilator induced blood flow8 and angiography9.

The first uncontrolled clinical trial of gene therapy for PAD in humans used a similar technique

The prospects for gene therapy in the management of peripheral vascular disease

An Viewpoint by Alex Rossdeutsch, UCL Institute of Child Health

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by treating patients with lower limb ischaemia with VEGF encoding naked plasmid DNA delivered via a catheter inserted through ulcerative lesions. Analysis showed marked de novo growth of collateral vessels as measured by MR and contrast angiography10. For the first time, this marked gene therapy as a viable potential strategy in PAD management. Subsequently, similar results were obtained following intramuscular injection of the plasmid - a necessary step forward as

the majority of patients with PAD do not display the ulcerative lesions required for catheter access11.

Following these small pioneering studies an array of phase II randomised placebo controlled double-blinded trials with predefined end-points have taken place to provide detailed analysis of the efficacy and safety of gene therapy strategies for PAD. Notable amongst these, a Finnish group dem-onstrated increased vascularisation as measured by

Fig.1: Summary of best practice management for the treatment of peripheral arterial disease (PAD). IC - intermittent claudication, CLI - critical limb ischaemia, ALI - acute limb ischaemia.

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digital subtraction angiography following treatment with VEGF using an adenoviral or plasmid/liposome vector infused during angioplasty12. One of the most important contributions of these trials was to establish the safety of gene therapy. Given that the field of gene therapy has been tarnished by the toxicity related death of a patient given adenovirus via the hepato-portal vein13 it was essential to establish the initial safety of research of this kind. Fortunately, it seems that there is no increased incidence of such theorised side effects as atherosclerosis, retinopathy or neoplasm14. Intramuscular plasmid VEGF therapy is associated with an increased peripheral oedema but this has been determined to have no significant effect on the health of the patient15.

However, there is a strong case for tempering enthusiasm with regards to the role of gene therapy in PAD, as a number of more recent and larger studies have reported negative results. The RAVE (Regional Angiogenesis with Vascular Endothelial growth factor) trial investigated exercise tolerance as described by the treadmill test time following intramuscular administra-tion of adenoviral encoded VEGF121 - an isoform of VEGF demonstrated to play a role in the initiation of angiogenesis16 and lumen formation in new vessels6. Analysis of this phase II trial revealed no significant improvement in patients receiving the intervention compared to controls17. It is noteworthy that trials of gene therapy for PAD have only yielded positive results when surrogate end-points such as angiogra-phy or quality of life measures have been used. The case for the potential of gene therapy in this regard is diminished by the recent publication of a study using a concrete end-point for the first time. Treatment of pa-tients with chronic limb ischaemia using intramuscular plasma encoded VEGF165 did not reduce the 100-day amputation rate when compared to controls18. Thus it could be said that strategies using gene therapy to treat PAD in their current form have only at best shown mild symptomatic relief but do not appear to positively affect the clinical outcomes of patients. To this end, further phase II/III trials could be undertaken to clarify this position using hard end-points such as amputation and surgical revascularisation procedure rates. This said, however, the best use of resources in the coming years might be better directed to optimising and improving methods of gene therapy, rather than continuing to repeat previous trials likely to continue in their production of equivocal results. Several lines of enquiry could be pursued in this respect.

It is quite possible that previous trials have

been hampered by low in vivo transfection efficien-cies. Measurements of transgene-containing plasmid uptake have been reported at levels of 5% and lower19. Adenoviral vectors display a similarly low infection rate20. Previously it has been proposed that a low trans-fection rate when treating PAD might not be so critical to the efficacy of the therapy as angiogenesis relies on the paracrine secretion of growth factors. Thus, it may be immaterial whether this is produced in large amounts by relatively few cells or at lower levels by many10. Duration of transgene expression also seems to be crucial. Former opinion in some sources held that treatment of PAD might be more acquiescent to gene therapy than other diseases as continued expres-sion of the transgene might not be a requirement for angiogenesis. After a vessel has been established, it was thought that growth factors such as VEGF would no longer be needed in a role of maintenance19. Re-cently however, experiments have been performed using a conditional switch for VEGF expression; al-lowing VEGF levels in the murine embryo to remain under endogenous control but adult VEGF levels to be controlled by exogenous administration of the growth factor. These indicated that neovascularisation could be accomplished entirely by VEGF but that a long duration of stimulus was required. If addition of the growth factor was stopped prematurely then the new vascular network would regress21. This indicates that vectors used in therapeutic angiogenesis might need to be longer acting than the traditional adenoviral and plasmid vectors used previously.

Many novel vector candidates are undergo-ing trials in animal models. Part of the reason for the poor transgene expression duration in adenovirus infected cells is thought to be due to an inflammatory response against the viral late proteins22. Use of a fully deleted helper dependent adenovirus is one possible means of overcoming this. Such an adenovirus has a genome which has been obliterated to remove any late protein expression and relies on a helper virus for its manufacture in vitro22. This type of "gutless" virus has been demonstrated to induce a sustained reporter transgene expression in mice and to overcome a pre-existing humoral anti-adenoviral immune response - a key attribute as it has been estimated that as much as 50% of the human population display this reactivity23. Another innovative line of attack is to use heterologous endothelial progenitor cells (EPC) transfected in vitro. Cells transduced in this manner, if selected for before re-implantation should display a 100% transfection rate. It has recently been shown that these cells can contribute to neovascularisation in mouse models and

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are not associated with the immune reaction normally viewed in conventional adenovirus infection24. Other potential vectors include adeno-associated viruses and non-viral liposomes used in conjunction with ultra-sound exposure to permeabilise target cells22.

It is important to realise that VEGF is only but a component in the large meshwork of protein signaling which contributes to angiogenesis. Studies are beginning to look at alternative angiogenic effector molecules as possible transgenes in the quest for more effective revascularisation. To this end clinical trials have taken place using fibroblast growth factor (FGF)25 and hepatocyte growth factor (HGF)26 but these suf-fer from the same constraints as previous trials with VEGF in that positive results are relatively undramatic and associated with the use of surrogate end-points. Avenues which might be more fruitful could be those which examine the therapeutic role of factors which can themselves induce a wide variety of angiogenic molecules in vivo. One example subject to study is hypoxia-inducible transcription factor (HIF)-1 which can stimulate a multitude of pro-angiogenic molecules such as VEGF, erythropoietin and IGF-213.

A variety of roles have been conjectured for the use of gene therapy in the context of therapeutic an-giogenesis. The hope of many was and still is that this approach can offer a front-line medical treatment for PAD without recourse to invasive surgical procedures - in this regard the field is now at a critical juncture. If the next generation of clinical trials featuring improved vector strategies and growth factors of increased po-tency cannot produce the evidence of concrete clinical benefit their forerunners lacked, then funding and opti-mism may decline. It appears unlikely that the world's healthcare providers will fund an expensive treatment that provides only moderate symptomatic relief and in most cases will only delay and not eliminate the requirement for surgery. In this respect, gene therapy also faces a resurgent challenge from therapy using recombinant protein where there is hope that expense is less and efficacy better than originally thought27. In spite of this pessimism however, there may yet be a niche for gene therapy to occupy. It is telling that the clinical studies with the most persuasive data have used surgical administration via catheter access for vector delivery. This raises the possibility that gene therapy could offer a prophylactic adjunct to surgery rather than a replacement of it. If gene therapy in the context of angioplasty versus angioplasty alone could be tested in a phase III clinical trial then even a relatively small improvement in the number of patients requiring fur-

ther costly surgery could well justify its use and prove the main task for gene therapy in the management of peripheral arterial disease.

References1. Dormandy, J. A., Rutherford, R.B. The TASC study group 2000. Mangament of peripheral arterial disease. Trans Atlantic Inter-Society Consensus (TASC). J. Vasc. Surg. 31, S1-S288 (2000).

2. Curci, J. A., Sanchez, L.A. Medical treatment of peripheral arterial disease. Current Opinion in Cardiology 18, 425-430 (2003).

3. Baumgartner, I., R., Schainfeld, L., Graziani. Management of Peripheral Vascular Disease. Annu. Rev. Med. 56, 249-272 (2005).

4. Yla-Hertualla, S., Martin, J.F.,. Cardiovascular Gene Therapy. Lancet 355, 213-222 (2000).

5. Morishita, R. Perspective in Progress of Cardiovascular Gene Therapy. J Pharmacol Sci 95, 1-8 (2004).

6. Carmeliet, P. Mechanisms of angiogenesis and arteriogen-esis. Nat. Med. 6, 389-395 (2000).

7. Ferrara, N. Role of Vascular Endothelial Growth Factor in the Regulation of Angiogenesis. Kidney Int. 56, 794-814 (1999).

8. Takeshita, S., Bauters, C., Asahara, T., Zheng, L.P., Rossow, S.T., Kearney, M., Barry, J.J., Ferrara, N., Symes, J.F., Isner, J.M. Physiologic assessment of angiogenesis by arterial gene therapy with vascular endothelial growth factor. Circulation 90, 1-90 (1994).

9. Takeshita, S., Zheng, L.P., Asahara, T., Riessen, R., Brogi, E., Ferrara, N., Symes, J.F., Isner, J.M. In vivo evidence of enhanced angiogenesis following direct arterial gene transfer of the plasmid encoding vascular endothelial growth factor,. Circulation 88, 1-476 (1993).

10. Isner, J. M. Arterial gene transfer of naked DNA for thera-peutic angiogenesis: early clinical results. Adv Drug Deliv Rev. 30, 185-197 (1998).

11. Baumgartner, I., Pieczek, A., Manor, O., Blair, R., Ke-arney, M., Walsh, K., Isner, J.M. Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia. Circulation 97, 1114-1123 (1998).

12. Mäkinen, K., Manninen, H., Hedman, M., Matsi, P., Mus-salo, H., Alhava, E., Ylä-Herttuala, S.,. Increased Vascularity Detected by Digital Subtraction Angiography after VEGF Gene Transfer to Human Lower Limb Artery: A Randomized, Placebo-Controlled, Double-Blinded Phase II Study. Molecular Therapy 6, 127-133 (2002).

13. Yla-Hertualla, S., Alitalo, K. Gene transfer as a tool to induce therapeutic vascular growth. Nat. Med. 9, 694-701 (2003).

14. Isner, J. M., Vale, P.R., Symes, J.F., Losordo, D.W. Assess-ment of Risks Associated With Cardiovascular Gene Therapy in Human Subjects. Circ. Res. 89, 389-400 (2001).

15. Baumgartner, I., Rauh, G., Pieczek, A., Wuensch, D., Magner, M., Kearney, M., Schainfeld, R., Isner, J.M. Lower-

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Extremity Edema Associated with Gene Transfer of Naked DNA Encoding Vascular Endothelial Growth Factor. Annal. Internal Med. 132, 880-884 (2000).

16. Carmeliet, P., et al. Impaired myocardial angiogenesis and ischemic cardiomyopathy in mice lacking the vascular endothelial growth factor isoforms VEGF164 and VEGF188. Nat. Med. 5, 495-502 (1999).

17. Rajagopalan, S., Mohler III, E.R., Lederman, R.J., Men-delsohn, F.O., Saucedo, J.F., Goldman, C.K., Blebea, J., Macko, J., Kessler, P.D., Rasmussen, H.S., Annex, B.H. Regional Angiogenesis With Vascular Endothelial Growth Factor in Peripheral Arterial Disease: A Phase II Randomized, Double-Blind, Controlled Study of Adenoviral Delivery of Vascular Endothelial Growth Factor 121 in Patients With Disabling In-termittent Claudication. Circulation 108, 1933-1938 (2003).

18. Kusumanto, Y. H., Van Weel, V., Mulder, N.H., Smit, A.J., Van Den Dungen, J.J.A.M., Hooymans, J.M.M., Sluiter, W.J., Tio, R.A., Quax, P.H.A., Gans, R.O.B., Dullaart, R.P.F., Hospers, G.A.P. Treatment with Intramuscular Vascular En-dothelial Growth Factor Gene Compared with Placebo for Patients with Diabetes Mellitus and Critical Limb Ischemia: A Double-Blind Randomized Trial. Hum. Gen. Ther. 17, 683-691 (2006).

19. Faries, P. L., Pomposelli Jr., F.B., Quist, W.C., LoGerfo, F.W. Assessing the Role of Gene Therapy in the Treatment of Vascular Disease. Annal Vasc Surg. 14, 181-188 (2000).

20. Elami, M., Gangadharan, S., Sui, X., Rhynhart, K., Sny-der, R., Conte, M. Gene delivery to in situ veins: differential effects of adenovirus and adeno-associated viral vectors. J. Vasc. Surg. 31 (2000).

21. Dor, Y., Djonov, V., Abramovitch, R., Itin, A., Fishman, G.I., Carmeliet, P., Goelman, G., Keshet, E. Conditional switching of VEGF provides new insights into adult neovas-cularization and pro-angiogenic therapy. The EMBO Journal 21, 1939-1947 (2002).

22. Khan, T. A., Selke, F.W., Laham, R.J. Gene therapy progress and prospects: therapeutic angiogenesis for limb and myocar-dial ischaemia. Gene Therapy 10, 285-291 (2003).

23. Maione, D., Della Rocca, C., Giannetti, P., D'Arrigo, R., Liberatoscioli, L., Franlin, L.L., Sandig, V., Ciliberto, G., La Monica, N., Savino, R. An improved helper-dependent

adenoviral vector allows persistent gene expression after in-tramuscular delivery and overcomes preexisting immunity to adenovirus. Proc. Natl. Acad. Sci. 98, 5986-5991 (2001).

24. Iwaguro, H., Yamaguchi, J., Kalka, C., Murasawa, S., Ma-suda, H., Hayashi, S., Silver, M., Li, T., Isner, J.M., Asahara, T. Endothelial Progenitor Cell Vascular Endothelial Growth Factor Gene Transfer for Vascular Regeneration. Circulation 105, 732-738 (2002).

25. Comerota, A. J., Throm, R.C., Miller, K.A., Henry, T., Chronos, N., Laird, J., Sequeira, R., Kent, C.K., Bacchetta, M., Goldman, C., Salenius, J.P., Schmieder, F.A., Pilsudski, R. Naked plasmid DNA encoding fibroblast growth factor type I for the treatment of end-stage irreconstructable lower extremity ischaemia: preliminary results of a phase I trial. J. Vasc. Surg. 35, 930-936 (2002).

26. Morishita, R., Aoki, M., Ogihara, T. Does gene therapy become pharmacotherapy? Exp. Physiol. 90, 307-313 (2005).27. Simons, M., Bonow, R., Chronos, N., Cohen, D., Giordano, F., Hammond, H., Laham, R., Li, W., Pike, M., Sellke, F.W., Stegmann, T., Udelson, J., Rosengart, T. Clinical trials in coronary angiogenesis: issues, problems, consensus. an expert panel summary. Circulation 102, e73-e76 (2000).

Alex Rossdeutsch is a MBPhD student

at the Moleular Medicine Unit,

UCL Institute of Child Health,

30 Guilford Street,

London WC1N 1EH

a.rossdeutsch@ucl.ac.uk

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http://www.bscr.org- Information on forthcoming meetings, workshops and symposia

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The principal objective of the meeting is to highlight current issues surrounding drug-induced torsades de pointes and will focus on biomarkers, risk factors and preclinical investigational methods. Furthermore, the meeting aims to allow individuals active in Industrial Safety Pharmacology to engage with clinical and non-clinical academic investigators. The programme will consist of three symposia incorporating presentations by leaders in the field followed by a moderated debate led by expert panel members.

BSCR Autumn Meeting 2007- Programme

"The QT interval and drug-induced torsades de pointes"Dates: 24-25th September, 2007

Venue: Governors' Hall, St. Thomas' Hospital, London

Organiser: Dr Michael Curtis

Day 1 - Monday 24th September12:30-1:50 Registration and buffet lunch1:50-2:00 Welcome and introduction

2:00-5:30 SYMPOSIUM 1: "Is QT prolongation always intrinsically arrhythmogenic, or intrinsically antiarrhythmic?" (with a tea interval from 4:00 to 4:30)

Participants:

Speaker 1 Luc Hondeghem Speaker 2 Dan Roden Expert panel member 1 Gary Gintant Expert panel member 2 Marc Vos Expert panel member 3 Jules Hancox Expert panel member 4 Russ Bialecki Expert panel member 5 Leif Carlsson Expert panel member 6 Rashmi Shah Chairman moderator Mike Curtis Rapporteur Antonio Cavalheiro

Two internationally recognised proponents of differing viewpoint (Speakers 1 and 2) will each present their case in a talk (30 min), then submit themselves to a panel of 6 experts who would lead a debate. Audience participation would be encouraged. A Chairman would moderate. A rapporteur would take minutes, and the whole event would be audiotaped for publication.5:30-7:00 Free communication (poster) presentation and assessment and wine reception

7:30 Conference dinner

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Day 2 - Tuesday 25th September9:00-12:30 SYMPOSIUM 2: "How validated are current models and biomarkers for testing drug-induced torsades de pointes liability?" (with a tea interval from 11:00-11:30)

Human volunteer phase 1 studies - Philip Sager The anaesthetized rabbit TDP model - Leif Carlsson The AV blocked canine preparation - Marc Vos QT interval and its corrections in the in vivo conscious canine - Anthony Fossa The rabbit heart failure model - Bob Hamlin The rabbit Langendorff preparation and the Screenit approach - Peter Hoffmann The wedge preparation - Gan-Xin Yan HERG screens - Jules Hancox

12:30-1:30 Lunch

1:30-4:30 SYMPOSIUM 3: "Drug-induced torsades de pointes - now what?" (tea 3:00-3:30)

Should preclinical TDP liability assessment take into account the influence of channelopathies? - Craig January

A drug for men and women - how important is gender as a TDP disposition risk factor? - Susan Coker

Inter-model comparisons - the experience of the QT Prodact initiative - Keitaro Hashimoto Are any of the preclinical TDP screens good for quantitative (dose-response) TDP liability

assessment? - Gary Gintant If a drug deemed 'safe' in preclinical tests subsequently prolongs QT in phase 1 studies how can its developer convince regulators to allow development to proceed? - Rashmi Shah Preclinical cardiovascular safety assessment - is this a career for new cardiovascular PhD

graduate? - Michael Pugsley

4:30-4:45 Prize giving and meeting close

Cardiovascular Related MeetingsAnnual Scientific Conference of the British Cardiovascular Society, 4th-7th June 2007 will be held in Glagow, UK. Further details can be obtained by visiting www.bcs.com and enquiries should be directed to enquiries@bcs.com

Heart Failure 2007 will take place in Hamburg, Germany, 9th-12th June. For further information, contact HFsecretariat@escardio.org

76th European Atherosclerosis Society Congress, 10-13 June 2007. Helsinki, Finland. For further infor-mation: Tel: +41 22 908 0488; E-mail: eas2007@kenes.com; Website: www.kenes.com/eas2007

XIX ISHR World Congress in Bologna, Italy 22-26 June 2007. Organizers Roberto Ferrari and Luigi Tavazzi. Enquiries: Prof. Roberto Ferrari, Chief of Cardiology, University Hospital of Ferrara, Corso Giovecca 203, 44100 Ferrara, Italy. E-mail: info@ishr-italy2007.org, Website www.ishr-italy2007.org

4th European Meeting on Vascular Biology and Medicine, University of Bristol, 17th-20th September, 2007. Visit the website: www.emvbm.org for refular meeting updates. Secretariat: Wheldon Events & Conferences. Tel:+44 (0)1922 457984; E-mail: info@wheldonevents.freeserve.co.uk.

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British Heart Foundation GrantsProject Grants Committee, November 2006Prof A D Hughes et al, Imperial College London. "Growth Related effects in ALSPAC on Cardiac Endpoints (GRACE) study" (3 years) £163,431 Dr C Emanueli et al, University of Bristol. "Role of Neu-rotrophin p75 receptor in the angiogenesis and apoptosis responses to limb ischaemia and cutaneous wounds in diabetic mice" (3 years) £151,473Prof J C Hancox & Dr H J Witchel, University of Bristol. "Comparative electrophysiology and pharmacology of mu-tant potassium channels in different variants of the short QT syndrome" (2 years) £83,897 Dr J C Mason et al, Imperial College London. "Protein kinase C epsilon - a regulator of cytoprotection against vascular endothelial injury" (3 years) £158,508Prof M R Bennett, University of Cambridge. "Apoptosis of vascular smooth muscle cells in vessel remodelling" (3 years) £192,705 Prof D A Eisner et al, University of Manchester. "Identifying how cellular calcium buffers modulate the systolic calcium transient and response to β-adrenergic stimulation in isolated cardiac myocytes" (3 years) £175,593Prof J P T Ward & Dr P I Aaronson, King's College London. "Enhanced pulmonary vascular reactivity in compensated hypercapnia: role of bicarbonate transporters and chloride channels" (3 years) £155,482Dr M J Morrell et al, Imperial College London. "Screening for sleep apnoea in chronic heart failure" (2.5 years) £157,422Prof M Perretti & Dr F D'Acquisto, Queen Mary University of London. "The impact of annexin 1 cleavage on neutrophil be-haviour during vascular inflammation" (3 years) £225,692Prof P H Whincup et al, St Georges, University of London. "Passive smoking, cardiovascular disease and Type 2 diabe-tes: prospective studies in older men and women" (1 year & 3 months) £145,086Dr A E Scott et al, University of Aberdeen. "Tissue deforma-tion echocardiography: a tool for risk stratification in hyper-trophic cardiomyopathy" (1 year) £11,050Prof D Wray, University of Leeds. "Disease-causing muta-tions in intracellular domains of the cardiac herg potassium channel" (1 year & 9 months) £79,634Dr C C Shoulders et al, Imperial College London. "Cloning the chromosome 8p23-22 cholesterol quantitative trait gene: crucial for ultimate dissection of the cholesterol trait of famil-ial combined hyperlipidaemia" (3 years) £224,152Prof K M Channon & Dr N J Alp, University of Oxford. "Endothelial cell repopulation and in-stent restenosis in novel mouse models" (3 years) £202,677Dr V Ohanian & Dr J Ohanian, University of Manchester. "A study of Hic-5 in vascular smooth muscle signalling" (3 years) £144,419 Dr S A Cook et al, Imperial College London. "Characterisation of microRNA in the heart" (3 years) £152,444 Dr J Willets et al, University of Leicester. "G protein-coupled receptor kinase (GRK) regulation of angiotensin II type 1and endothelin A receptor-mediated smooth muscle excitability" (3 years) £155,679

Fellowships Committee, January 2007Senior Clinical Research Fellowship Dr I A Wilkinson, University of Cambridge."Identification of the mechanisms responsible for large artery stiffening in man" (5 years) £973,519

PhD Studentships Miss J D Clarke, University of Manchester. "Mechanisms underlying altered calcium homeostasis in the atria in heart failure" (1 yr 9 months) £53,891 Miss C Pierides, University of Surrey. "Characterisation of the effect of immunisation with apoB-100 derived peptides on the proliferation and functional properties of regulatory T cells" (3 years) £81,587 Unnamed and Clerk, Imperial College London. "Rho family small G proteins in cardiac myocyte hypertrophy and death (3 years) £97,751 Unnamed and Nourshargh, Imperial College London."Role of PECAM-1 in cytokine- & chemokine-induced leukocyte transmigration and extravascular motility in vivo: a comparative study with JAM-A (3 years) £99,077 Unnamed and Heath, University of Birmingham. "The role of RhoJ/TCL in angiogenesis" (3 years) £88,818 Miss N M Campbell, St George's, University of London. "Salt and blood pressure in children, a cross-sectional study and an intervention study (3 years) £93,649 Miss S Lake, Kings College London. "The role of Gci1 in human platelet activation" (3 years) £96,809

Intermediate Clinical Research FellowshipsDr J J Boyle, Imperial College London."Does cd163 regulate an antioxidant transcriptional program in culprit atheroscle-rotic lesions?" (4 years) £549,475

Clinical Research Training FellowshipsDr R C Shroff, University of Cambridge. "An in-vitro model of intact human arteries to study the mechanisms of vascular calcification in chronic kidney disease: clinical and laboratory correlation" (2 years) £138,487 Dr R Sofat, University College London. "Is human comple-ment factor H a shared risk factor for age-related macular degeneration and atherosclerosis?" (3 years) £179,103 Dr G V Rowlinson, Imperial College London."Disorders of connexins in congenital heart disease" (3 years) £210,795 Mr J R Finch, Imperial College London. "NF-KappaB activity and inhibition in vein graft accelerated intimal Hyperplasia" (2 years) £458,870

MBPhD Studentship Mr A Rossdeutsch University College London (ICH). "Investi-gating the role of thymosin β4 during coronary vessel develop-ment and neovascularisation" (2 yrs 9 months) £88,022

Project Grants Committee January 2007Dr I J Mackie, University College London."The modes of inhibition of factor Xa by heparin in plasma: neutralisation by platelet factor 4" (1 year) 47,921 Prof J E Deanfield et al, University College London (ICH). "Genetic and environmental determinants of arterial function in childhood: insight into causal pathways from the Avon Longitudinal Study of Parents and Children (ALSPAC)" (3 years) £240,286

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Dr H M Arthur & Prof B D Keavney, University of Newcastle."Circulating endothelial progenitor cells: investi-gation of the role of vascular injury and cardiac aging in two patient cohorts" (2 years) £114,789Dr B J Wojciak-Stothard et al, University College London. "ADMA/DDAH pathway as a critical regulator of endothelial motility and blood vessel growth" (2 years) £94,114Prof D A Lane, Imperial College London. "Characterisation of the molecular basis of protein S enhancement of tissue factor pathway inhibitor (TFPI) anticoagulant function" (3 years) £167,330Dr G F Clough et al, University of Southampton. "Develop-mental dietary mismatch and gender in the aetiology of endo-thelial dysfunction in metabolic syndrome: the mechanistic role of oxidative stress" (2 years) £93,476Dr Z Shui & Prof M R Boyett, University of Manchester. "Role of cholinergic stimulation and pulmonary vein sleeves in atrial fibrillation" (3 years) £202,567Dr M Emerson, Imperial College London. "Endothelial and platelet derived nitric oxide as distinct mediators of platelet function in vivo" (2 years) £97,543Dr S Kennedy et al, University of Glasgow. "Cardioprotection and the modulation of vascular tone by anandamide is medi-ated by sphingosine 1-phosphate" (2 years) £83,704Prof D P Taggart et al, University of Oxford."A high-flow acoustic filtration technique to remove lipid microemboli from blood" (3 years) £159,701Prof D J Beech et al, University of Leeds. "Functions of STIM and Orai proteins in vascular smooth muscle cells" (3 years) £192,093Dr S Kennedy et al, University of Glasgow. "Modulation of calcium handling mechanisms in healthy and atherosclerotic vascular smooth muscle" (3 years) £142,563Dr B Latinkic, Cardiff University. "Molecular dissection of cardiogenic activities of GATA4" (3 years) £188,093 Dr K K Ray et al, University of Cambridge."Large scale

studies of metabolic risk factors in coronary heart disease" (1 year) £89,050Prof A H Baker et al, University of Glasgow."Does ACE2 have a protective role in the heart? Systematic analysis of ACE2 in a disease model" (3 years) £172,483Prof C N McCollum et al, University of Manchester."The role of venous to arterial circulation shunts, cerebral emboli and endothelial dysfunction in migraine" (2 years) £204,881Dr N Mills et al, University of Edinburgh."Endothelial pro-genitor cells in acute vascular injury and repair" (3 years) £242,685 Dr A Clerk, Imperial College London. "Kruppel-like factors 2, 4 and 6 in cardiac myocyte hypertrophy" (1 year) £58,142

Dr H M Arthur & Prof B D Keavney, University of Newcastle upon Tyne. "Endothelial progenitor cells and angiogenesis: the role of TGF?" (3 years) £149,964 Dr P J R Barton et al, Imperial College London."Role of the calcineurin splicing variant CnAβ1 in heart failure and recovery" (3 years) £174,043Prof S E Harding, Imperial College London."Maturation of human and mouse embryonic stem cell-derived cardiomyo-cytes in culture and after implantation into the heart" (2 years) £119,146 Dr A M Randi et al, Imperial College London."Role of the transcription factor Erg in inflammation" (3 years) £204,506

Chairs and Programme Grants Committee February 2007Infrastructure GrantProfessor G D Angelini, University of Bristol. "Equipment for the Bristol Heart Institute" £150,000

Programme GrantProfessor M J Shattock et al, King's College London. "Regula-tion of the cardiac Na/K ATPase in health and disease: role of phospholemman (FXYD1)" 5 years £1,249,302

Cardiovascular Related Wellcome Trust GrantsAugust to October 2006

Wellcome Programme GrantProfessor David J Paterson, University of Oxford. Heart Physiome 37 Months £89,747Programme GrantsProfessor Richard J Evans, University of Leicester. Characteri-sation of P2x1 Receptor Function, Regulation and Develop-ment of Molecular Models of Drug Action at ATP Gated P2x Receptors 60 Months £1,128,406Professor Sussan Nourshargh, Imperial College School of Medicine, London. An Investigation into the Molecular and Cellular Mechanisms Involved in Mediating Neutrophil and Monocyte Transmigration In Vivo. 60 Months £901,654Dr A Mark Evans, University Of St Andrews. Is AMP-Acti-vated Protein Kinase Sufficient and Necessary for Calcium Signalling by Hypoxia in Oxygen-Sensing Cells? 60 Months £1,033,134Research Career Development FellowshipDr Jana Barlic, Imperial College London. Mechanisms of Atherosgenesis: How Oxidized Low Density Lipoprotein, its Derivatives and Nuclear Receptors Regulate Expression and Function of Pro-Atherogenic Chemokines and Chemokine

Receptors 60 Months £662,508Research Training FellowshipsDr Eoin F Mckinney, Cambridge Institute for Medicine Re-search. A Prospective Investigation of Gene Expression Profil-ing in Primary Systemic Vasculitis 36 Months £242,463Dr Houman Ashrafian, University of Oxford. The Role of Cardiac Stem Cells in Response to Cardiac Injury. 36 Months £203,960Dr Omolola O Ayoola, University of Manchester. The Effect of Maternal Health, Fetal Size and Early Childhood Growth on Cardiovascular (CVS) Development in Nigerian Children. 48 Months £426,810Project GrantsDr Timothy M Frayling, University of Exeter. Molecular Ge-netic Studies in Hypertension, Type 2 Diabetes, and Coronary Heart Disease in Pakistan. 36 Months £285,239Professor Martin Griffin, Aston University. Control of Central Venous Catheter Obstruction and Infection by Selective Inhibi-tion of Transglutaminase. 36 Months £226,014Professor David H Adams, University of Birmingham. The Role of Vascular Adhesion Protein-1 in Leucocyte Transmigra-

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BSCR Autumn Meeting 2007THE QT INTERVAL AND DRUG-INDUCED

TORSADES DE POINTESDates: Monday 24th and Tuesday 25th September, 2007

Venue: Governors' Hall, St Thomas' Hospital, London, UK

Organiser: Michael J Curtis PhD

Objectives: The principal objective of the meeting is to highlight current issues surrounding drug-induced torsades de pointes and will focus on biomarkers, risk factors and preclinical investigational methods. Furthermore, the meeting aims to allow individuals active in Industrial Safety Pharmacology to engage with clinical and pre-clinical academic investigators.

Programme: The programme consists of three symposia. The first is a moderated debate, led by expert panel members, and is entitled "Is QT prolongation always intrinsically arrhythmogenic, or intrinsically antiarrhythmic?" The second and third symposia are entitled: "How validated are current models and biomarkers for testing drug-induced torsades de pointes liability?" and "Drug-induced torsades de pointes - now what?". The finalized list of speakers and panel members is: Luc Hondeghem, Dan Roden, Philip Sager, Russ Bialecki, Leif Carlsson, Marc Vos, Anthony Fossa, Bob Hamlin, Peter Hoffmann, Gan-Xin Yan, Jules Hancox, Craig January, Susan Coker, Keitaro Hashimoto, Gary Gintant, Rashmi Shah, Michael Pugsley.

Travel & Accommodation: Attendees are requested to make their own arrangements for travel and accommodation. UK and mainland European attendees should require accommodation for the night of Sept 24th only. The meeting location is 5 minutes walk from Waterloo mainline station, Waterloo Underground, and Westminster Underground stations. Therefore hotels advertised on the web as located near any station on the, Bakerloo, Circle, District, Northern, or Jubilee underground lines will provide convenient access to the venue.

Free communications: Free communications are by poster. There are two poster prizes of £250 each: the Clinical Science Young Investigator Award and the BSCR Young Investigator Award (you must be a BSCR member to enter).

Registration (excluding accommodation): Free for BSCR members and £40 for academic non-members.

Bursaries: The Society will consider awarding travel grants of up to £200 per person to bona fide students who are members of the BSCR.

The full programme, abstract pro-forma, meeting registration form, and student bursaries application form are available for downloading from the BSCR website (www.bscr.org).

The deadline for the submission of abstracts, registration and application for student bursaries is 10th August 2007. Early registration is recommended owing to a limit of 150 persons for the meeting, and 100 for the conference dinner.

Further enquiries: Enquires regarding the programme, registration or accommodation should be directed to Mr Antonio Cavalheiro, Cardiovascular Division, Rayne Institute, St Thomas' Hospital, London SE1 7EH; Tel: +44 (0) 207 1881095; Fax: +44 (0) 207 1883902; E-mail: Antonio.cavalheiro@kcl.ac.uk.

Enquires regarding BSCR membership or student bursaries should be directed to Prof. Barbara McDermott, BSCR Secretary, Therapeutics & Pharmacology, Queen's University Belfast, Whitla Medical Building, 97 Lisburn Road, Belfast BT9 7BL; Tel 02890-972242; Fax 02890-438346; b.mcdermott@qub.ac.uk