ICIN. united minds for better hearts. Annual Report 2007

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1 ICIN united minds for better hearts Annual Report 2007

2 Directors W.H. van Gilst, PhD E.E. van der Wall, M.D. PhD Scientific Council E.E. van der Wall MD, PhD (Leiden) chairman W.H. van Gilst, PhD (Groningen) co-chairman A.A.M. Wilde, MD, PhD (Amsterdam) A.C. van Rossum, MD, PhD (Amsterdam) D.J. van Veldhuisen, MD, PhD (Groningen) E.E. van der Wall, MD, PhD (Leiden) H.J.G.M. Crijns, MD, PhD (Maastricht) F.W.A. Verheugt, MD, PhD (Nijmegen) M.L. Simoons, MD, PhD (Rotterdam) P.A. Doevendans, MD, PhD (Utrecht) and ICIN Professors: J.M.T. de Bakker, PhD I.C. van Gelder, Md, PhD W.J. van der Giessen, MD, PhD J.W. Jukema, MD, PhD C.L. Mummery, PhD Y.M. Pinto MD, PhD A.C. van Rossum, MD, PhD A.F.W. van der Steen, PhD International Scientific Commiittee Prof.dr. W.J. Paulus (voorzitter), VUMC, Amsterdam Prof.dr. P.A. Poole-Wilson, Imperial College London Prof.dr. G. Breithardt, Universitätsklinikum Münster Prof.dr. Th.F. Lüscher, Universität Zürich Prof.dr. M. Komajda, Hôpital Pitié-Salpétrière, Parijs Dr. M. Konstam, Tufts-New England Medical Center, Boston Staff General management: Sjoukje Holtrop (until Oct 2007) & Jan Weijers (since Nov 2007) Financial management: Arnold Heijkamp Personnel: Femke Meerdink Veldboom (until November 2007) Reception: Corry Schouten Finance: Judith Thijzen The Interuniversity Cardiology Institute of the Netherlands PO Box DG Utrecht The Netherlands The Interuniversity Cardiology Institue of the Netherlands is an institute of the Royal Netherlands Academy of Arts and Sciences.

3 Contents Introduction & 68: Ultrasound contrast agents : GENetic DEterminants of Restenosis (GENDER) : Molecular basis of inherited cardiac diseases : The ICTUS studies & 57: Atheroexpress : Neointimal Hyperplasia and Endothelial Function : Congenital heart disease in adults : Mechanisms And Treatment Of Tachyarrhythmias : Cardiac Development and Genetics : In vivo mouse and rat mri and mrs : Shear stress and the vulnerable plaque : Segmentation of the moving left ventricle : MRI imaging : Delivery through ultrasound and microbubbles : Implications MSCT findings for subsequent management : RACE II : DPTE : HEBE trial : PRIMA study & 21: Special ultrasound transducers : HEBE III Trial : Biopacemaker : Drug-related sudden cardiac death : In search of genes for cardiac conduction : Immunath : Right ventricular dysfunction and tetralogy of Fallot : Arrhythmogenic Right Ventricular Dysplasia : Developmental biology of the heart : Careful ICIN Publications in Summary financial report

4 Introduction 4 The year 2007 was a moving year with sad and glorious moments. A tragic moment was the untimely death of Prof. dr. Cees Visser, January 27th, 2007, Director ICIN from His impressive death ceremony February 3rd was attended by numerous colleagues, relatives and friends. Prof. Visser had been a passionate Director of the ICIN and he was already extensively commemorated in our Annual Report As of the first of March 2007, Prof. dr. Ernst van der Wall formally took over the position of Prof. Visser. Together with Prof. dr. Wiek van Gilst, they will be the ICIN Directors for the forthcoming years. A glorious moment was the 35-year celebration of the ICIN June 6th in the Barbizon Hotel Amsterdam. A special halfday symposium on behalf of the 7th lustrum of the ICIN was organized, and the meeting was dedicated to Prof. dr. Cees Visser. The meeting was well attended and we had excellent speakers such as Prof. dr. F.P. van Oostrom (the president of KNAW, the Royal Netherlands Academy for Arts and Sciences), Prof. dr. F.M. Meijler (former ICIN Director), Prof. dr. M. J. Janse (emeritus professor AMC), Prof. dr. Y. M. Pinto (ICIN-professor), Prof. dr. H.J.G.M. Crijns (President NVVC) and dr. T. Opthof (AMC). The keynote lecture was given by Prof. dr. K.A.A. Fox (UK) entitled: Translational research: how to improve care? For this special occasion a jubileum book was published entitled 35 jaar interuniversitaire cardiologie in Nederland Time for Reflection (Editor Mrs. M. Helmers, former general manager of ICIN). We would like to acknowledge all individuals who have contributed to this special occasion. At the spring meeting of the Netherlands Society of Cardiology (NVVC) in April 2007, the ICIN/NVVC Einthoven dissertation prizes were awarded to Mrs. Dr. M.R.M Jongbloed (first prize), Mr.dr. E. Lipsic (second prize), and Mrs.dr. P.S. Monraats (third prize). A memorable moment was the launch of United Hearts, a unique platform consisting of 4 main cardiovascular institutions/ organizations/bodies in the Netherlands; Netherlands Heart Foundation (NHS), Netherlands Society of Cardiology (NVVC), Patient Union (SHHV), and the ICIN. This unified platform has the primary aim to guarantee the continuity and quality of prevention, patient care, and cardiovascular research in the near and distant future. United Hearts should also be the common voice-tube to the political and public arena. July 7th (070707) we had our first combined meeting in Leuvenum to set common goals and to define priorities in the cardiovascular domain in the Netherlands. A second Leuvenum-meeting was organized November 17th in order to streamline and fine tune the ideas of the first meeting resulting in four scientific spearheads; 1) congenital heart disease, 2) heart failure, 3) cardiac arrest, and 4) stroke. It was well recognized that atherosclerosis may be the common underlying mechanism in the majority of these disease entities. At the end of the second Leuvenum meeting the Treaty of Leuvenum was officially ratified and undersigned by the 4 involved parties (NHS, dr. H. Stam; NVVC, Prof.dr. H.Crijns; SHHV, Mrs M. Weerts; ICIN, the two Directors). It is sincerely hoped for that United Hearts will be seen as the major cardiovascular entity in the Netherlands both by the community and the government. On 4 April and 8 October ICIN held two project days. These days offer a platform to young researchers to present their work. A jury judges the presentations and awards prizes. In April the prize was awarded to Joanne Schuijf for her presentation MSCT in the non-invasive evaluation of coronary artery disease. In October two prizes were awarded, one to Arie van Erk for his presentation Genomics Program: Virtuous intentions, malign consequences and one to Carianne Verheugt for Prognosis of adults with CHD in the CONCOR national registry. In October 2007 we took formal leave of our general manager Mrs. dr. S. Holtrop who decided to follow an other pathway in her career. Of course we like to thank her for all her efforts dedicated to the ICIN and we wish her success in her personal and professional life. As of November, J. Weijers was appointed as new general manager of the ICIN and we hope for a longstanding relationship with ICIN. ICIN united minds for better hearts In late 2007 ICIN developed a new website and got a new logo. The eight red dots in the new logo together form the heart and they also symbolise the eight university medical centres. The MagICINe was completely redesigned and so were our other publications. Interuniversity Cardiology Institute of the Netherlands

5 5 In December we had our yearly Projectronde in order to review and select the proposals we had received in the fall. Traditionally this is being done by our Scientific Council, the ICIN-professors and external reviewers consisting of non-project involved experts in the Netherlands. A new aspect of reviewing our projects was the installation of a KNAW-instigated international Scientific Committee consisting of Prof.dr. W.J Paulus (VUmc, Amsterdam: Chairman), Prof.dr. P.A. Poole-Wilson (London, UK), Prof.dr. G. Breithardt (Muenster, DE), Prof.dr. T.F. Luesher, Zurich, CH), Prof.dr. M. Komajda (Paris, FR) and Prof.dr. M. Konstam (Boston, USA). The international Scientific Committee will act as an additional objective layer to select the most excellent projects and to assist in defining new strategies. We had received 16 proposals of which 8 passed the first evaluation round. In total 5 projects (2 of these for 50%) were awarded after careful judgment by the different reviewing bodies. The following projects were awarded ICIN funding: Angiotensin II receptor blockers in patients with a systemic right ventricle Prevention of atrial fibrillation: markers for stretch and pacing induced structural remodeling and the preventive efficacy of upstream therapy Human cardiomyocyte progenitor cells: novel in vitro model for inherited cardiac disease Biomechanics of vulnerable plaque rupture Sudden cardiac death in the young (1-40 years of age): occurence, causes and the yield of cardiogenetic screening in surviving first degree relatives In 2007, ICIN awarded two Fellowships. Rolan van Kimmenade will work with dr Januzzi of the Massachusetts General Hospital in Boston and dr DeFilippi of the University of Maryland Medical Centre in Maryland on the CAPRICORN-CRS study, the problems of vascular calcification in heart failure patients with a reduced kidney function.. Daniel Pijnappels will go to the Cardiovascular Research Center, Harvard Medical School, Boston to work on Electrophysiological determination of cardiomyocytes obtained from adult fibroblasts by genetical modification. ICIN professor Ton van der Steen was appointed Simon Stevin Master by the Dutch research organisation STW. Throughout the year, we had our monthly assemblies of the Scientific Council consisting of the 8 heads of the Department Durrer center for cardiogenetic research of Cardiology, the 8 ICIN-professors and additional expert ICIN project leaders. In addition, two very interesting project days were organized which allow the exposure of scientific progress within the major ICIN projects. At the research level, the intake in the HEBE 1 trials went successful and we are looking forward to hear the final results in Along those lines, the Cardiogenetic Research Center (CRC) was further developed and renamed as Durrer Center. The official launch of the Durrer Center is expected in the spring of Lastly, we have strengthened our relationships with the NHS, the NVVC and the Working group Cardiovascular research the Netherlands (WCN) in order to promote the field of cardiovascular research in the Netherlands. ICIN s redesigned web site The scientific output of our institute continues to grow. As you can see on the final pages of this report, in 2007 the collective efforts of our researchers resulted nearly in 300 publications in the top journals of the world. In addition, ICIN researchers produced 60 meetings abstracts. 13 researchers obtained their doctoral degree in Overall, the number of staff of ICIN remained constant in comparison to By the end of 2007, the secretariat had 7 positions, three of which were vacant (the vacancies have been filled now). 83 people worked for ICIN in scientific positions, partly on our payroll and partly posted with us by academic hospitals. ICIN is an institute of KNAW, the Royal Dutch Academy for Arts and Sciences. Government funding is given to ICIN through KNAW. KNAW also provides services to ICIN in the fields of legal affairs, ICT and personnel. We thank the KNAW for providing financial means and administrative backup, the Hubrecht institute for hosting three of our research groups, the Scientific Council for assisting us in taking the right decisions and setting out new strategies, and the ICIN secretariat (Arnold Heijkamp, Jan Weijers, Judith Thijzen, Eelco Soeteman, Rachida Tallahi, Marjan de Jonge en Corry Schouten-Schonewille) for their continuous support and important contribution to the success of ICIN. Ernst E. van der Wall, MD, PhD Wiek. H. van Gilst, PhD Directors of ICIN Annual Report 2007

6 21 & 68: Ultrasound contrast agents 6 General In project 21 ultrasounds contrast agents (small encapsulated bubbles) are studied for clinical diagnostic and therapeutic use. These projects are co-sponsored by SenterNovem, STW, FP6 program, and ICIN. Companies directly participating in this research are Philips Medical Systems, Bracco, and Oldelft. In the Netherlands the Universities of Wageningen (WU), Twente (UTwente), Groningen (UMCG), and Amsterdam (VU) are also directly involved in these projects. Conference As the years before the Twelfth European conference on ultrasound imaging was very successful. As the time of writing the Thirteenth lie already behind us (evenly successful) and we are already preparing for the Fourteenth conference, to be held January 22nd and 23rd, 2009 near a wall is also coupled with its acoustic image (see Figure). It is thus important to study the competition between the secondary Bjerknes force and the binding force of a functionalized bubble with the receptor. For this part of the study, the optical tweezers was used to position the bubbles UCA microbubbles (BR-14, Bracco Research S.A., Geneva, Switzerland) away from the sample chamber wall prior to insonation. This prevents sliding friction at the wall and decouples the acoustic effect due to the wall vicinity, allowing a full quantification of purely the effect of the bubble-bubble interaction. Two optical traps (see Figure) were generated and the ultra-high speed camera Brandaris128 was used to optically record the bubble dynamics with sub-μs time resolution. Bubble behaviour: Mode vibrations Normally the vibrations of bubbles in an ultrasound field are symmetrical. Under certain condition related to the frequency and amplitude of the ultrasound field the bubble can start to vibrate asymmetrical and showing a so-called mode vibration. The lowest order is a mode 2 but we have noticed mode vibration up to order 5. In the figure below a mode 3 is shown, which represent itself as a varying triangular shape during the ultrasound wave. Mode vibrations are important for subharmonic imaging and for enhanced drug delivery with the aid of bubbles Mode-vibrations as recorded with the Brandaris128. Bubble size: 4 µm. Ultrasound frequency 3.5 MHz. Recording speed: 10 million frames per second. Left: bubble at rest. Right: Bubble showing Mode 3 vibration. Binding force An optical tweezers setup is being developed to study the full force balance on targeted UCA microbubbles, including binding interaction with the target, viscous drag due to the blood flow, acoustic radiation forces (primary and secondary Bjerknes forces) arising from the applied sound field. Bubbles are acoustically coupled to each other through the secondary Bjerknes force to neighboring bubbles, and a bubble In the ultrasound field, a targeted UCA microbubble experiences a secondary Bjerknes force (Fbj) due to the vicinity of a second bubble, but also due to its acoustic image when it is close to a wall. The effect on the ligand-receptor binding (Fmol) needs to be quantified for the two components. The effect of viscous drag (FD) is also important in the design of targeted bubbles. In the ultrasound field, a targeted UCA microbubble experiences a secondary Bjerknes force (Fbj) due to the vicinity of a second bubble, but also due to its acoustic image when it is close to a wall. The effect on the ligand-receptor binding (Fmol) needs to be quantified for the two components. The effect of viscous drag (FD) is also important in the design of targeted bubbles. The peak values of the instantaneous secondary Bjerknes force found in our experiments on two bubbles of 3-μm radius, initially 11 μm apart, are of the order of up to 10-6 N. This value is to be compared with the typical average value (order or 10-8 N - typically, only the (time) averaged value of the secondary Bjerknes force is known from experiments). The primary Bjerknes force in the same conditions is of the order of N. The same setup was used to study the occurrence of surface modes on UCAs when any influence of a neighboring wall was excluded by positioning them 50 µm away from the wall. Interuniversity Cardiology Institute of the Netherlands

7 7 24: GENetic DEterminants of Restenosis (GENDER) Introduction Percutaneous coronary intervention (PCI) has become a safe and effective treatment modality for single and multivessel coronary artery disease. However, restenosis is still the major limitation of PCI, resulting from injury of the vessel wall caused by balloon dilation and stent placement. The vascular damage is characterized by irritation of endothelial and subendothelial structures and injury of medial regions with rupture of the internal elastic lamina. This damage causes segmental thrombus formation and subsequent invasion of macrophages and polymorphonuclear leukocytes, followed by expression and release of numerous growth factors and cytokines from blood cells and stretched smooth muscle cells, leading to proliferation of smooth muscle cells. Vascular inflammation thus plays an important role in this complex multifactorial phenomenon. Restenosis is a major health problem, because it occurs in 10-40% of all treated persons, depending on which technique was used and the extension of the disease. No pharmacologic strategy or new device has proven effective in preventing this phenomenon. Only coronary stenting and the upcoming drugeluting stents (DES) have reduced the incidence of restenosis, but certainly did not abolish it. In return difficult to treat in-stent restenosis might develop. Identifying patients at increased risk for restenosis may improve stratification of patients to individually tailored treatment. Thus far, however, it has proven difficult to stratify patients with regard to risk for coronary restenosis based only upon clinical or procedural risk factors, since risk factors identified so far in relation to restenosis have not been consistently reported. There is evidence that genetic factors explain part of the excessive risk for restenosis independently of conventional clinical variables. Therefore, the purpose of this study was to evaluate in a large unselected study sample whether a variety of inflammatory genetic determinants can predict the risk of clinical restenosis after PCI. The GENDER project The GENetic DEterminants of Restenosis project was designed to study the association between various gene polymorphisms and clinical restenosis. It is a multicenter prospective followup study with both clinical and angiographic restenosis as an endpoint. All patients undergoing percutaneous coronary interventions were eligible for the study. Only, patients treated for acute ST elevation myocardial infarction (MI) were excluded. After having obtained written informed consent, blood was sampled for DNA isolation and future analysis. Clinical and procedural data were gathered prospectively. Clinical restenosis was established during a 9-month follow-up for death, myocardial infarction and target vessel revascularisation. A repeat angiographic study was performed in a subpopulation after 6 months. An independent endpoint committee evaluated all potential endpoints. As mentioned before, inflammatory responsiveness, which is highly genetically determined, plays a pivotal role in the process of restenosis. Therefore, we concentrated on inflammatory genes. We mostly selected genes, on basis of their relation to inflammation previously described in literature reports of gene associations with inflammatory diseases. Participating centers Participating centers in the GENDER project are the Leiden University Medical Center in Leiden, the Academic Medical Center in Amsterdam, the University Hospital Maastricht and the University Medical Center Groningen. The Gaubius Laboratory of TNO-PG in Leiden collected the blood samples from the participating centers and isolated DNA. DNA-analysis was executed in the University Hospital of Maastricht and in the Sylvius Laboratory Leiden. Furthermore, we cooperated with the Erasmus University of Rotterdam to analyze several other candidate genes. The mouse model experiments were performed at the Gaubius Laboratory of TNO-PG. Present status Table 1: Baseline characteristics Total (n=3104) Age (years) 62.1 ± 10.7 BMI (kg.m-2) 27.0 ± 3.9 Male sex 2216 (71.4 %) Diabetes 453 (14.6%) Hypercholesterolemia 1890 (60.9%) Hypertension 1259 (40.6%) Current smoker 762 (24.5%) Family history of MI 1098 (35.4%) Stable angina 2079 (67.0%) Multivessel disease 1432 (46.1%) Lipid lowering medication 1687 (54.3%) Restenotic lesions 208 (6.7%) Total occlusions 428 (13.8%) Type C lesion 802 (25.8%) Proximal LAD 689 (22.2%) In 1998 the GENDER project was designed. Approval of the Medical Ethics Committees of the participating centers was obtained. Inclusion of patients started in March In June 2001 the last patient was included in the project. DNA has been extracted for all patients and genotyping for 114 SNPs in different genes has been performed by using two different arrays (inflammation and cardiovascular), developed by Roche Molecular Systems. Furthermore, seven different multiplex assays, with more than 100 different polymorphisms, divided over 25 different genes have been developed and executed at the Sylvius laboratory. Among these genes were EP300, CREBBP and PCAF, epigenetic genes encoding histone acetyltransferases (HATs), which are known to have a major influence on gene expression. Four other polymorphisms have been analysed by Taqman analysis also at the Sylvius laboratory. In cooperation with the Erasmus University we genotyped 15 polymorphisms in the Vitamin D-receptor gene. Furthermore, we used an established mouse model of restenosis to study the involvement of the significantly associated genes with restenosis. This was Annual Report 2007

8 8 Gene (polymorphism) 95% CI HR Low High P-value CSF2 Ile117Thr ADRB2 Arg16Gly CD C/T Eotaxin G/A ApoA4 Gln360His ApoC C/T ApoE Arg158Cys LPL Ser447Ter TNFα -238 G/A TNFα T/C PAI1 4G/5G ins/del F5 Arg506Gln AGTR 1166 A/C MMP9 99 G/A Caspase G/A IL G/A IL G/A IL A/G Vit D receptor G/C Vit D receptor A/G Vit D receptor -25 C/A Vit D receptor 464 G/T PCAF G/C Marburg I (G534E) Quaking C/T Quaking 2786 T/C Quaking A/C Quaking A/G Quaking A/G Quaking A/G Quaking A/G performed in cooperation with the Gaubius Laboratory of TNO- PG. And we performed ELISA s in lipopolysaccharide whole blood to determine the functionality of some of the polymorphisms we found to be significantly associated with restenosis. Recently, we finished the genotyping of several polymorphisms in different candidate genes and gene systems, which can lead to an even better understanding of the restenotic process and thereby improve risk stratification of patients even more. These new analyses include polymorphisms in matrix metalloproteinase genes and in a number of other genes involved in vascular remodelling, such as the hyaluronan binding protein (HABP2), cathepsin S (CTSS) and galectin 3 (LGALS3). In addition, we analyzed several genes which have not been implicated in cardiovascular disease before, including the Quaking gene and a few histone deacetylases (HDACs), epigenetic proteins which are known to counteract HATs. Cardiologist, Present results Total follow-up was completed in 3,146 patients, after exclusion of patients who developed an event in the first 30 days, 304 (9,8%) patients developed restenosis (target vessel revascularization, TVR). Angiographic follow-up is available in 480 patients. Indeed, so far, we were able to identify 31 polymorphisms in 19 different genes associated with restenosis. Eight of these genes have already been validated in an established mouse model of restenosis, where they showed time-dependent upregulation and could thus confirm a role for these genes in the restenotic process. After analysis of the epigenetic genes, a polymorphism in the promoter of the PCAFgene showed an association with both clinical and angiographic restenosis. We already confirmed its significance in another large prospective study (the PROSPER-study) and at the moment we are further investigating its functional significance using EMSAbandshift assays. The recent analysis of the Quaking gene showed strong associations of polymorphisms in the promoter region and intron 3 of this gene with TVR. The role of Quaking in restenosis is currently being investigated in an animal model. These findings contribute to the unravelling of the process of restenosis. Furthermore, genotyping of these genes may provide a better risk stratification and lead to a more tailored therapy for patients with identified increased risk of restenosis after PCI and may serve as rationale for new anti-restenotic therapies. Participating investigators Principle investigators Prof. Dr. J.W. Jukema (project leader), LUMC/ICIN Scientific committee Dr. R.J. de Winter, Cardiologist, AMC Prof. Dr. P.A.F.M. Doevendans, Cardiologist, UMCU Dr. R.A. Tio, Cardiologist, AZG Prof. Dr. A. van der Laarse, Biochemist, LUMC Prof. Dr. R.R. Frants, Anthropogenetica, LUMC Mw.Dr. M.P.M. de Maat, Biochemist, EUR Prof. Dr. A.H. Zwinderman, Statistician, AMC Prof. Dr. J. Waltenberger, Cardiologist, AZM Prof. Dr. E.E. van der Wall, Cardiologist, LUMC Study coordinator Dr. P.S. Monraats, Research fellow, ICIN/LUMC Drs. D. Pons, Research fellow, ICIN/LUMC Interuniversity Cardiology Institute of the Netherlands

9 9 27: Molecular basis of inherited cardiac diseases The ICIN project 27 focusses on the Molecular basis of inherited cardiac diseases associated with arrhythmias. The project aimed to identify disease-causing genes and to unravel the pathophysiological basis of inherited cardiac diseases. This line had no new financial input in the past years and as in 2006 the remaining publications resulted from work started in previous years. The focus shifted further on different cardiomyopathies, among which hypertrophic cardiomyopathy (1,2) and dilated cardiomyopathy (3,4), although research in the primary arrhythmia syndrome field was also still succesful (5). In particular, a new type of mutation in the human ryanodine receptor type II (hryr2) was described leading to a unique phenotype (6) The GENCOR database, installed early 2006, is steadily growing and has now over 800 patients from 7 (academic) centres included. As in 2006, newsletters kept the participants up to date. The half-yearly meetings with all cardiologists, interested in genetics, and clinical and molecular geneticists from all university centers continued to be a platform for discussions in this rapidly evolving field. One of the direct results of these meetings is a guideline for genetic testing in hypertorphic cardiomyopathy, the first of its kind worldwide. Genomics Programme Project 50 is a closely related project where genomic techniques are used to identify crucial novel mechanisms in the development of cardiac hypertrophy. The project encompasses both basic and clinical genetic studies, focusing on mouse, rat and human cardiac hypertrophy. The first phase of the project focused on identification of specific genes whose altered expression heralds the progression from apparently compensated cardiac hypertrophy towards overt heart failure. This has now become scientifically productive as one of the major papers has been published in 2007 in a high ranking journal (7). This paper describes the identification of a novel component of the intercalated disc LIMP-2. These studies also have led to a very productive collaboration with dr. Stuart Cook and prof. Timothy Aitman, from the Imperial College of London, UK. Our groups joined their efforts to integrate genomic (expression data) with genetic strategies (gene mapping in rat inbred strains). This has yielded a completely novel molecule important for the development of LVH, findings that have now been accepted for publication in Nature Genetics The human genetics part of the program is now well underway, aided by the impressive collection of human HCM subjects that has been built up. We anticipate to produce significant results from the human genetics part in the coming two final years of this program. Annual Report 2007

10 29: The ICTUS studies 10 The ICTUS studies are a collaboration between ICIN and WCN and a large number of Dutch cardiologists and address the question what is the optimal revascularization strategy in high risk patients with an acute coronary syndrome but without STelevation on the electrocardiogram. Between 2001 and 2003, a total of 1200 patients non-ste-acs with an elevated cardiac troponin T were randomized in one of the 42 participating hospitals between an early invasive and a selective invasive strategy against a background of optimized medical therapy. The main results have been published in the New England Journal of Medicine in In 2007, the prospectively planned long-term follow-up was published in the March 10th issue of the Lancet. In summary, the main results of ICTUS showed that there was no mortality reduction associated with an early invasive strategy, neither at one year nor at four years. The results of the ICTUS study have been included in several meta-analyses concerning treatment strategies in nste-acs that were published in 2006 and additional analyses concerning female patients and patient with Diabetes Mellitus have been presented at the American Heart Scientific Meetings in Orlando in collaboration with Michelle O Donoghue and Eugene Braunwald of the Brigham and Womens Hospital, Boston, USA. Importantly, the results of ICTUS have been incorporated in the updated 2007 AHA / ACC guidelines on diagnosis and management of patients with non-ste-acs. A selective invasive strategy has been given a class II, level of evidence B, recommendation for medically stabilized patients, including patients with an elevated cardiac troponin. Although the updated guidelines of the European Society of Cardiology did non include such a recommendation, the results of the ICTUS studies are included in the analyses and accompanying text by the guidelines writing committee. Several substudies as underway and papers have been published or are in preparation. The prognostic value of peripheral blood levels of NT-proBNP on admission in ICTUS has been published in the American Heart Journal An ICTUS substudy regarding the value of ST-segment changes and outcome has been published in the Journal of Electrocardiology A cost-analysis has been published in January 2008 in the International Journal of Cardiology. The association between revascularization status at discharge and long-term mortality compared to the randomized treatment allocation will be presented at the American College of Cardiology Scientific Meetings in Chicago Project leader, Dr R.J. de Winter has contributed a review regarding invasive therapy in non- STE-ACS for Harrison s principles in Internal Medicine, On-line edition Several other biomarker substudies are underway, including the value of GDF-15 and outcome, renal (dys-)function compared to levels of Cystatin C and outcome, and a number of markers of inflammation. There are now three large randomized trials that have collected long-term follow up: the ICTUS study, the FRISC-II study and the RITA-3 study. A collaboration has been initiated with prof Lars Wallentin from Uppsala, Sweden (principal investigator of the FRISC-II study) and prof Keith Fox, Oxford, United Kingdom (principal investigator of the RITA-3 study) to perform meta-analyses and subgroup analyses of the three trials combined on a per-patient basis. The senior statistician of this collaboration is Prof. J.G.P. Tijssen from the AMC. Plans for the future are several subgroup analyses regarding the association between gender and long term outcome, biomarker status including level of cardiac troponin and long term outcome, differences in risk regarding procedure-related myocardial infarctions and spontaneous myocardial infarctions. An international collaboration is underway to organize a large prospective randomized clinical trial to determine the optimal treatment strategy in women with non-ste-acs. Figure 1: Cumulative risk of death (upper panel) or death or spontaneous myocardial infarction (lower panel) from hospital discharge to end of follow-up in patients randomized to an early invasive strategy. The presence of revascularization at discharge is associated with an approximately 50% reduction in long term mortality in retrospective cohort Landmark analysis. However, randomization to an early invasive strategy was not associated with long term outcome. The explanation is that the non-revascularized patient population consists of patients without significant coronary artery disease and patients with severe three-vessel disease and serious co-morbidities that carry a very high risk of revascularization procedure related complications (and thus have a poor prognosis). Patient were divided into 3 groups: (1) patients that were revascularized during initial hospitalization, (2) patients that were not revascularized because angiography did not show a significant coronary stenosis of 70% ( 50% for left main) (group A), and (3) remaining patients that were not revascularized because of other reasons (group B). Abbreviations: Revasc, in-hospital revascularization; CAD, coronary artery disease Interuniversity Cardiology Institute of the Netherlands

11 11 33 & 57: Atheroexpress Karlijn van Keulen, funded by the Dutch Heart Association and OIO since August 2004, is studying Toll-like receptor signaling in arterial remodeling. Arterial remodeling is the process of a structural change in size of the artery, which can be outward or inward remodeling. During atherosclerosis arterial remodeling occurs, inward arterial remodeling accelerates luminal narrowing by atherosclerotic lesion formation, while outward arterial remodeling compensates for luminal stenosis. Outward remodeling seems to be a favorable process, but is related to unstable atherosclerotic lesions, prone to rupture which leads to clinical symptoms like heart or cerebral infarction. She uses the carotid artery ligation model for outward remodeling. The right carotid is ligated, and the blood flow in the left carotid artery increases and this will induce outward arterial remodeling. To get outward remodeling toll-like receptor 4 (TLR4) plays a crucial role, which has been shown in our lab using the ligation model. Now we are investigating proteins upstream and downstream of TLR4 to get more insights in this pathway during arterial remodeling. in TLR4 KO mice) but we observed even a decrease in luminal area of the arteries. We are investigating the precise role of EDA in arterial remodeling using microarray data and validation methods like qpcr. The first results of this study are presented on the DAS 2007 and Karlijn received a DAS fellowship award for this presentation. She hopes to publish the results of this study in Downstream of TLR4 transcription factor NF-kappaB is activated. Last year, Karlijn investigated the role of the NF-kappaB p50 subunit in outward remodeling. This p50 subunit is the inhibiting subunit of NF-kappaB signaling. In p50 knockout mice this brake is absent, and we indeed observed more pronounced outward remodeling (measured as external elastic lamina area = EEL) in p50 KO animals. These results are written down and send in for publication. Next year, Karlijn will finish the currently ongoing studies and write her thesis. Wouter Peeters PhD student since November 2006 funded by the EU Wouter determined in more then 500 human atherosclerotic plaques,11 pro- and anti-inflammatory cytokine levels. The cytokine data were associated with the Atheroexpress clinical and histological data. This showed that rupture-prone (vulnerable) plaques contain much more proinflammatory cytokines then stable plaques. Next to this, he found that shortly after an ischemic event (stroke, TIA) there is strong decrease in proinflammatory cytokines that is independent of anti-inflammatory medication. These data are presented at the AHA in Orlando and are reported in an original paper that has been submitted. These findings give new insights that are relevant for the analysis of new biomarkers in local atherosclerotic plaques that are predictive for future cardiovascular events. Fig 1 Increase in External Elastic Lamina Area (EEL) as a measure of arterial size in NfkB p50 null mice after ligation of the contralateral carotid artery Upstream we look at endogenous ligands of TLR4. Extra domain A (EDA) of fibronectin is one example of such an endogenous TLR4 ligand. In 2007 Karlijn [published the article Levels of extra domain A containing fibronectin in human atherosclerotic plaques are associated with a stable plaque phenotype in the journal Atherosclerosis (Atherosclerosis Nov;195(1):e83-91). In a healthy artery EDA is not expressed, but in atherosclerotic tissue EDA expression is found around macrophages, endothelial cells and smooth muscle cells. EDA is also involved in atherogenesis in mice. In the Atherosclerosis article she described the relation between plaque EDA levels and stable atherosclerotic plaques, containing high collagen levels with high smooth muscle cell content. Concomitantly, asymptomatic patients also show higher EDA plaque levels compared to symptomatic patients. EDA plasma levels did not correlate with plaque phenotype or clinical symptoms. The role of EDA in arterial remodeling is studied in EDA knockout mice. EDA KO mice not only show less outward remodeling (like Another project focusses on neovascularisation and thrombus formation in the atherosclerotische plaque. Within this project plaque blood vessels and thrombus are carefully analysed and associated with plaque phenotype and clinical data. Furthermore Wouter is working on the validation of new biomarkers that were identified in the proteomics study in Singapore. Focus is on markers that have a predictive value for future cardiovascular events. Arjan Schoneveld Head Technician since July 1995 Next to taking care of the organization of the laboratory of experimental cardiology in Utrecht and helping others with their research, Arjan was able to conduct the following study: Atherosclerotic lesion development and Toll Like Receptor 2 and 4 responsiveness. Both Toll like receptors (TLR) and their endogenous ligands have been well recognized for their role in atherosclerotic lesion development. Since repetitive stimulation of TLR induces an attenuated inflammatory response, we hypothesized that the TLR response is altered during atherosclerosis development, due to chronic exposure to endogenous ligands. He examined aging mice between 5-40 wk (both ApoE-/- and C57Bl/6). In ApoE-/- mice with advanced of atherosclerosis, levels of mrna Annual Report 2007

12 12 encoding the TLRs as well as the endogenous TLR ligands were increased. This showed that systemic TLR cell surface expression on circulating monocytes and levels of endogenous ligand in the plasma were increased in aging atherosclerotic mice. We also observed that endogenous TLR ligands were capable of activating white blood cells through their respective TLR2 (MFI) TLR2 C57Bl/6 age (wk) TLR2 ApoE-/- TLRs. During the plaque progression however, stimulation of TLRs in blood samples demonstrated attenuated cytokine responses in atherosclerotic mice. This showed that, although TLR expression increases in atherosclerotic plaques and on circulating cells, monocytes became less responsive to TLR activation, which may be due to chronic TLR engagement by endogenous ligands. Dominique de Kleijn Project leader (Funded by the EU) is focusing on the validation of the biomakers he elucidated in Singapore on an outgoing EU grant. These results identify a new concept that local atherosclerotic TLR4 (MFI) TLR4 C57Bl/6 age (wk) TLR4 ApoE-/- Fig 2 TLR expression on circulating blood monocytes. Flow cytrometric analysis demonstrating the difference in expression of TLR2 (A) and TLR4 (B) between C57Bl/6 control mice and ApoE-/- mice over time. Data is represented as mean fluorescence intensity (MFI). Each datapoint depicts the mean ± SD.* significant difference between C57Bl/6 and ApoE at indicated timepoint (p<0.05, Mann-Whitney). plaque contain predictive molecular information of future cardiovascular events. Next to this, he continues working on the role of Toll-like receptors in cardiovascular disease but shifts now more towards the role in the heart after myocardial infarction. This is also the focus of the role of Mesenchymal Stemcell conditioned medium in collaboration with Singapore that has a protective effect on infact size after reperfusion injury. He is also the program leader of the CTMM LipidMap program investigating the role and application of local plaque lipids and serum lipids for the prediction of cardiovascular events. Athero-Express. The aforementioned study athero-express is a biobank study with a longitudinalm study design. About 1600 patients undergoing carotid endarterectomy [n=950], femoral enarterectomy [n=350] or aortic surgery [n=300] are now included. All patients fill in questionnaires and undergo follow up. In this study we search for local plaque biomarkers that have a predictive value for vascular events originating in all vascular territories. This approach appears very successful. The first follow updata revealing recently discovered biomarkers showed that local plaque proteins hide prognostic information for the occurrence of clinical events in the entire vascular system with strong predictive power. This biobank is also used in several interuniversity collaborations. DNA telomere lengths will be assessed from 900 patients who were included by Pim van der Harst [UMCG]. In addition other collaboratons are being discussed with partners within the UMCG and other Dutch UMCs and Universities elsewhere in Europe. Interuniversity Cardiology Institute of the Netherlands

13 13 34: Neointimal Hyperplasia and Endothelial Function Brachytherapy Brachytherapy has been the focus of a study dealing with the long-term effects of PCI. This entailed studying both the acute and chronic effects of beta-radiation on proliferation, inflammation and tissue growth in porcine arteries. Figure 1 illustrates the acute effect (3 days) on the proliferative response. Especially at the site of an arterial rupture, expression of PCNA (proliferating cell nuclear antigen) is enhanced (Fig. 1). Radiation tends to decrease the proliferative response at these sites (Fig. 2). Figure 3. Overview (top left) and detail of an artery at two years following brachytherapy (20 Gy). Note the red stained collagen denoting medial fibrosis and the thickened adventitia (top left, arrow) New techniques such as OCT (optical coherence tomography) can easily detect these changes in vivo (Figure 4, 5). Figure 1. Overview (top left) and detail of a control artery 3 days following PCI, stained for PCNA and showing a proliferative response at the site of injury Figure 4. Histology (L) and in-vivo OCT (R) of an internal Iiliac artery. Irradiation results in medial thickening and severe adventitial fibrosis (small side branch in 4 o clock position). Figure 2. Overview (top left) and detail (R) of an irradiated artery (20 Gy) at 3 days following PCI. Proliferation is less intense. The chronic or late effects of radiation were studied at two years following brachytherapy. The effects are clearly seen in the media and the adventitia. Both tissue layers show an enormous increase in collagen deposition, with the media being partly fibrosed (Figure 3). Fig 5. Normal porcine iliac artery (L) and iliac artery two years after beta irradiation (R) showing negative remodeling and fibrosis Annual Report 2007

14 14 Gene therapy research Intracardiac gene delivery with the use of recombinant Semliki Forest virus (SFV). Previously, SFV was shown to be a highly efficient vector for in vitro gene delivery in cardiovascular myocytes (Roks et al. 1997, 2002). The past year, SFV was tested in vivo in the infarcted rat heart, where it was delivered by means of the clamping technique. SFV was abundantly expressed in the noninfarcted myocardium for more than one week (Fig. 6). SFV was only expressed in the heart, whereas adenoviral vector led to expression in both heart and liver. SFV is a promising vector for myocardial gene therapy. The results will be published in Journal of Molecular and Cellular Cardiology, 2004 by Loot et al. (1-7) infusion reduces the development of heart failure after myocardial infarction in rats (Loot et al. 2002). In 2003, the physiological role and therapeutic potential of Ang-(1-7) were further explored. The physiological function of Ang-(1-7) as an antagonist of Ang II was tested after low and high sodium diet in rats. A low sodium diet led to the dissapearance of the non-competitive antagonism of Ang II, whereas after a high sodium diet this function of Ang-(1-7) was maintained (Fig. 7). The NO-independent, noncompetitive inhibition of Ang II constrictions was mediated by AT2 and Ang-(1-7) receptors. The results suggest that during a low sodium diet endogenous Ang II inhibitors, such as Ang-(1-7), are attenuated to prevent hypotension through salt deprivation. The therapeutic potential of Ang-(1-7) with respect to its inhibitory effect on vascular smooth muscle cell proliferation are being evaluated in the newly developed rat in-stent restenosis (ISR) model. Coronary stents are being implanted into the rat abdominal aorta to measure ISR after 4 weeks of treatment with saline or Ang-(1-7). Typical parameters for ISR such as neointimal area will be measured. Cardiomyoplasty Fig 6. (left) LacZ expression in the rat heart (black spots in left ventricle) after SFVlacZ administration by means of (right) the clamping Renin-angiotensin system in cardiovascular disease Angiotensin-(1-7) function further explored. Cell therapy of the infarcted myocardium can be performed by autologous cells. Crude bone marrow is one of the options and can be easily obtained. It has recently been associated however with a more intense inflammatory response at the infarct site. Well defined myocardial infarctions can be made in chronic pig models by a two hour inflation of a PTCA balloon, followed by reperfusion. In these experiments crude bone marrow was injected directly into the coronary artery at one week following MI, at the site of prior occlusion. Angiotensin-(1-7) (Ang-(1-7)) is a hormone that counterregulates Ang II effects. It has antihypertensive effects, protects against myocardial reperfusion damage and acts antiproliferative on vascular smooth muscle cells. We previously showed that it is a non-competitive antagonist for Ang II vasoconstriction in (human) arteries independently from stimulation of nitric oxide (NO) production (Roks et al. 1999). Furthermore, chronic Ang- contraction (% of PE-max) Effect of Ang-(1-7) on rat aortic Ang II response after salt diet log [Ang II] (mol/l) high sodium control high sodium Ang-(1-7) low sodium control low sodium Ang-(1-7) Figure 8. Contrast MRI (delayed enhancement) showing both delayed enhancement of the reperfused but infarcted myocardium ( between arrows) as well as an area showing no-reflow (L, baseline). The right hand panel shows delayed enhancement of a thinned wall at 5 weeks follow-up. MRI techniques such as delayed enhancement for infarct size measurements (Fig. 8), global LV-mass, -volume and -ejection fraction as well as regional functional measurements( Fig. 9) can be performed both at baseline (1 week following MI) and at follow-up (5 weeks following MI; Figs. 10 and 11). These functional measurements are then followed by histological assessment. Interuniversity Cardiology Institute of the Netherlands

15 15 RV LV Figure 9. Segmentation of the LV wall in a short-axis view. This is used to calculate wall thickening both at baseline and follow-up green = normal wall; pink = anterior borderzone; green = infarct zone; blue = posterior borderzone Figure 11. End diastolic volume (EDV) as measured by MRI, showing an increase in EDV in all groups representing normal growth. MI animals show a larger EDV as compared to sham (no MI) as the result of dilatation. There is no significant effect of bone marrow injection on EDV. Figure 10. Representation of baseline (L) and follow-up (R, grayscale) wall thickening of (left to right) anterior border, infarct, posterior border and remote myocardium (n=3, ± SEM). Participants: W. van Gilst (RUG/ICIN), M. Harteveld (ICIN), A. Moelker (EMC), D. Duncker (EMC), H. van Beusekom (EMC), A. Roks (RUG), D. van Essen (NHS), A. Loot (ICIN), Marx (ICIN), Boddeus (ICIN), R.Tio (RUG), W. Kerver (EMC), I. Peters (EMC), R-J. van Geuns (EMC), W. van der Giessen (EMC). Collaboration: Dept Cardiological Radiology EMC; Dr. Doris Taylor, Duke University, Durham, USA; David Kolstad, Lightlab Imaging Inc. Westford, USA; Dr. Stephan Wnendt, Kourion Therapeutics AG, Langenfeld, Germany; Prof. Sylvia Bradamante, University of Milano, Italy Annual Report 2007

16 36: Congenital heart disease in adults 16 In 2007, eight studies were running on long term outcome of congenital heart disease ( Eisenmenger syndrome, Marfan syndrome, Transposition of the great arteries, Tetralogy of Fallot, Quality of Life, Gender differences and prognosis, Aortic Coarctation and Down Syndrome) and 2 studies on the genetic basis of congenital heart disease (Zebrafish and genes for heart development,and Euro Heart Repair). These studies resulted in 20 articles published in international peer reviewed journals in The CONCOR registry, a national registry and DNAbank for patients with CONgential CORvita (www.concor.net) had an important scientific spinoff in 2007: 27 scientific projects were using the CONCOR database and 30 articles have been published since its initiation in The number of patients included in CONCOR has been increased up to In 2007 drs M.G.Duffels coordinated the project as ICIN investigator and dr.e.t. van der Velde gave his support as ICT consultant. Three nurses, Lia Engelfriet, Irene Harms and Sylvia Busken were travelling along the 102 participating centers, both university and non-university hospitals. Cardiac screening of Down patients in an institution Angiontensin II receptor blockers Drs. M.M. Winter continued his ICIN project on the effect of angiontensin II receptor blockers in patients with a systemic right ventricle (RV). Although mid-term survival is relatively good in this patient group, morbidity is common, and evidence based therapeutic options are lacking. His study, a prospective, randomized trial, aims to fill this lack of knowledge for this young patient group. Six academic hospitals participate in his study. As follow-up after treatment initiation is three years, final conclusions cannot yet be drawn. However, baseline investigations have already lead to three oral presentations at international conferences and the submission of three articles to peer reviewed journals. Most frequent main diagnoses of 8600 adult patients with congenital heart defects in CONCOR. Prognosis of patients Drs. C.L. Verheugt has been working on her project on the prognosis of patients with congenital heart disease. In the CONCOR registry, gender differences have been found in the risk for complications. Women have a 33% higher risk of pulmonary hypertension, a 33% lower risk of aortic complications, a 47% lower risk of endocarditis, and a 55% lower risk of ICD implantation. Moreover, the CONCOR database has recently been linked to data from RVthe national registries on mortality (CBS) and on morbidity and hospital admissions (LMR). The analyses on mortality and morbidity will be performed throughout Congenital heart disease in patients with Down syndrome Drs. J.C. Vis continued his ICIN project on congenital heart disease in institutionalized patients with Down syndrome. He performs a cardiac screening program to optimize treatment of Down syndrome patients with a congenital heart defect. Furthermore, patients from institutions with a congenital heart defect will be included in the CONCOR registry. Four chamber Magnetic Resonance Image of a patient with a transposition of the great arteries after an atrial switch operation. Note the abundant trabeculations in the dilated and hypertrophied systemic right ventricle. LV = left ventricle; RV = right ventricle Interuniversity Cardiology Institute of the Netherlands

17 17 Pulmonary hypertension in adults Drs. M.G.J. Duffels continued her ICIN project on pulmonary hypertension in adults with congenital heart disease. Over 4% of the patients registered in the CONCOR registry are known to have pulmonary hypertension, mostly due to a (previous) leftto-right shunt. The Eisenmenger syndrome, the extreme form of pulmonary hypertension, with irreversible increased pulmonary vascular resistance, reversal of the shunt and cyanosis was found within 1% of all patients. Bosentan, an endothelin receptor antagonist, has been shown to reduce pulmonary arterial pressures in patients with pulmonary hypertension. So far, 60 patients with pulmonary hypertension due to congenital heart disease are treated with bosentan, following a standardized treatment protocol. Preliminary results are promising; bosentan is well tolerated, exercise capacity and clinical status seem to improve and no major adverse events have occurred. This nationwide study is performed in collaboration with the University Medical Center Groningen and the University Medical Center Nijmegen. Eligible patients can be included from all Dutch hospitals. High dose statins in post-coarctectomy patients In september 2007, drs. G. Mackay started the ICIN project High dose statins in post-coarctectomy patients. In this multicentre trial, the effect of the use of statins on atherosclerosis in adult post-coarctectomy patients is investigated by measuring Intima Media Thickness (IMT) at baseline and three years follow up. Preliminary results will be presented at the NVVC and ESC congress. Magnetic Resonance angiogram (3D reconstruction) in a patient with Aortic Coarctation ( arrow) Echocardiographic apical 4-chamber view in a patient with an atrioventricular septal defect (x) and the Eisenmenger syndrome. LV= left ventricle, RV= right ventricle, LA= left atrium, RA= right atrium. Annual Report 2007

18 38: Mechanisms And Treatment Of Tachyarrhythmias 18 Ventricular arrhythmias and sudden cardiac death. A non-invasive modality to determine pro-arrhythmic mechanisms in primary sodium channelopathies Background: The current study concerns the Brugada syndrome, which is electrocardiographically characterized by a distinct ST-segment elevation in the right precordial leads. Patients with spontaneously occurring Brugada ECG have a high risk for sudden cardiac death. ST-segment elevation in Brugada syndrome is often dynamic or concealed, but can be provoked by sodium channel blockers. Two major types of ST-segment elevation in the right precordial leads are distinguished. Type 1 Brugada syndrome is characterized by a coved ST-segment elevation > 0.2 mv and followed by a negative T-wave. Brugada syndrome is definitely diagnosed when a Type 1 ST-segment elevation is observed in more than one right-precordial lead (V1-V3) in conjunction with documented arrhythmias and a family history of SCD. Type 2 ST-segment elevation has a saddleback appearance with ST-segment elevation > 0.2 mv and followed by either a positive or biphasic T-wave. Conversion by application of cardiac sodium channel blockade of Type 2 STsegment elevation to Type 1 is also diagnostic for the Brugada syndrome. Originally, Brugada syndrome has been associated with mutations in the gene encoding for the cardiac sodium channel. However, SCN5A mutations occur only in 18-30% of Brugada syndrome cases. Data from heterozygous SCN5A mice and explanted human hearts from patients with a sodium channel mutation suggest that structural abnormalities, induced by the channel mutation, are an important component of Brugada syndrome that determines its arrhythmia vulnerability. of pharmacological intervention and selective ablation of areas with abnormal (structural) tissue characteristics. 4) To derive specific syndrome linked ECG features in overlap syndrome. Results: These involve results from 3 sub-studies. 1. Carto-Mapping Electro-anatomical CARTO mapping of the right ventricular endocardium has been carried out in Brugada syndrome (Br-S) patients and controls (supraventricular tachyarrhythmias). Three groups of patients were studied: (1) BrS-1 patients, i.e., BrS patients (diagnosis according to the consensus criteria) with a type-1 BrS-ECG during CARTO mapping (n=8). Two of these patients had a type-2 BrS-ECG at baseline and a type-1 BrS-ECG was only induced upon ajmaline provocation test; in these patients, RV CARTO mapping was conducted both before (type-2 BrS-ECG) and after ajmaline provocation (type-1 BrS- ECG); (2) BrS-2 patients, i.e., BrS patients with a type-2 BrS-ECG during CARTO mapping (n=9, not including the two patients in whom a type-1 BrS-ECG was induced by ajmaline provocation); (3) controls, i.e. patients who underwent electrophysiologic study for atrioventricular nodal reentrant tachycardia or lone atrial fibrillation (n=9); these patients had no history of syncope or SCD, or family history of SCD. Structural heart disease was excluded by magnetic resonance and/or echocardiographic imaging. The following results were obtained: (1) in BrS-1 patients, RV activation duration was 23 ms Hypothesis: Our original hypothesis was that computer simulation of sodium channel mutations is able to reveal ECG patterns that are related to the impaired ion channel characteristics and/or induced structural changes. Our computer simulations so far show that a pure sodium channel mutation that reduces current by up to 70% is unable to generate ST-segment elevation of either Type 1 or Type 2. Simulation of scattered fibrosis in the outer 50% of the RV wall had only minor effects on the QRS configuration and no ST-segment elevation. However, reduction of the sodium current in this model resulted in coved type ST-segment elevation in the precordial leads V1-V3. STsegment elevation was based on functional conduction block and electrotonically mediated. This also shows that the model is able to reveal the effect of pharmacological intervention, which is our second hypothesis. The third hypothesis that the model will be able to reveal specific features of overlap syndromes is still to be proven. Study objectives: 1) to determine the major pro-arrhythmic parameters (ion channel defect and/or structural abnormalities) in individual patients with Brugada syndrome by patient tailored computer simulation and CARTO mapping. 2) to determine whether the pro-arrhythmic parameters can be extracted from characteristics in 12 lead ECG or BSM. 3) To simulate the effect Figure 1. Three-dimensional reconstructions of (A) activation and (B) fractionated electrograms in antero-posterior view of a Brugada type 1 patient. Activation starts in the RV-apex (red) and diverges toward the outflow tract (purple). Duration of activation is significantly prolonged (94ms) with respect to controls (65 ms). Panel B shows an increased and patchy distribution of fractionated electrograms throughout RV. longer than in controls (ANOVA p<0.001) (Figure 1). In contrast, BrS-2 patients and controls had similar RV activation duration. (2) fractionation of endocardial electrograms was significantly higher (21%, ANOVA, p<0.001) in BrS (type 1 and 2) patients than in controls. (3) Heart rate corrected activation recovery intervals (ARIc) as a measure of action potential duration (APD) was significantly (ANOVA, p<0.001) shorter in both BrS groups than in controls (by 54 ms (mean value) in BrS-1, and by 44 ms (mean value) in BrS-2), but not different between BrS-1 and BrS-2 patients. In conclusion, the CARTO data show that Brugada syndrome patients displaying a Type-1 Brugada- ECG have pronounced conduction slowing, (2) there are more fractionated electrograms in Brugada syndrome patients with either a Type-1 Brugada ECG or a Type-2 Brugada ECG, and (3) activation recovery intervals as a measure of action potential duration are shorter in both BrS-1 and BrS-2 patients. Interuniversity Cardiology Institute of the Netherlands

19 19 2. Animal studies and an explanted human heart We tested our hypothesis that the preferential right ventricular involvement in Brugada syndrome is caused by a different structural and functional organisation of myocardium and the conduction system. a. Different structural organization: We observed fractionated electrograms at the epicardium of the RVOT in isolated, Langendorff-perfused pig hearts during septal stimulation in 8 out of 8 animals, but never at the right ventricular free wall. Upon histological analysis a sub-epicardial layer with a different fiber orientation was detected in the right ventricular outflow tract. Currently we are testing the effect of flecainide on the amount of fractionation in this area. b. Heterogeneous response to sodium channel blockade (Purkinje fibers): We investigated whether Purkinje fibres are less sensitive than ventricular myocardium to activation delay by cardiac sodium channel blockade due to a higher peak sodium density in Purkinje fibres than ventricular cardiomyocytes. This would lead to preferential activation delay in regions functionally devoid of Purkinje fibers. This was tested in the hearts of three open-chested pigs by analysing the activation delay of Purkinje spikes and local myocardial activation before and after flecainide (1.2 mg/kg iv). Purkinje activation at each identified site was less delayed by flecainide than myocardial activation (16 +/- 5 % vs. 36 +/- 9 %; mean±sem; p < 0.04; n = 3). Flecainide induced the largest activation delay at the latest activated, basal regions of the heart, including the RVOT. This caused an increase in QRSduration from 53 to 73 ms (p < 0.01; n = 3) with preservation of the initial part of the QRS-complex. c. Heterogeneous response to sodium channel blockade (structural loss of cell-to-cell connections): We hypothesized that an heterogeneous structural loss of cell-to-cell connections combined with a reduction of the sodium current leads to current-to-load mismatch conditions, functional conduction block and Brugada-type ST-segment elevation. We tested this hypothesis in the explanted, Langendorff-perfused heart of a 16-year old, female patient with dilated cardiomyopathy and a loss-of-function mutation in the cardiac sodium channel. The patient underwent cardiac transplantation for end-stage heart failure. A pseudo-ecg was recorded by immersing the heart in a container filled with perfusion fluid and fitted with three electrodes. Whole myocardial mapping was performed using 194 electrodes evenly distributed over the endocardium and epicardium of both ventricles. At baseline, a single short-coupled extra stimulus at the septum was followed by new fractionated activation at more right than left endocardial electrode positions (38% vs 6%, p < 0.001). After ajmaline, ST-segment elevation of 0.3 mv was observed in pseudo-avr. Local electrograms did not show sufficient late activation, nor sufficient early repolarisation throughout the ST-segment to be a cause of the elevation. Monophasic ST-segment elevation was observed on local electrograms from the electrodes on the basal epicardial right ventricle. The local contribution to the ST-segment elevation was confirmed by the Laplacian technique. These data show that right precordial ST-segment elevation can be caused by functional block of conduction due to current-to-load mismatch condition in right-sided structural heart disease. 3. Computer Model The computer model of Potse/Gulrajani has been adapted to meet the requirements for the current study: incorporation of an RVOT, regional differences in ion currents. Tools to make regional (electrophysiological, structural) changes in the model have been developed. Globally reduced sodium channel expression by 50% in combination with high expression of transient outward current was implemented in the model to simulate Brugada syndrome. Although action potentials at the right ventricular epicardium showed extremely deep notches, no ST-segment elevation was observed in the calculated surface ECG. When sodium channel expression was reduced to 30%, QRS widening occurred. QRS width doubled at a current expression of 10%. Even then, no Brugada ECG signs were found. We conclude from this sub-study that reduced sodium current alone cannot explain the ECG features. Subsequent simulations were inspired by the electrophysiologic findings in an explanted heart of a young patient suffering from severe dilated cardiomyopathy in the presence of an SCN5A mutation. Intracardiac recordings in this heart demonstrated low-amplitude electrograms, delayed activity and ST-segment elevation on epicardial local electrograms of the right ventricle after ajmalin. We tried to reproduce these phenomena of the local electrogram with the computer model by introducing an area of increased resistance between the cells and in the interstitium. Although the calculated ECG during sinus rhythm showed large epsilon waves in the right precordial leads, a Brugada ECG type was not obtained. Based upon the RV histology of the explanted heart which revealed extensive sub-epicardial fibro-fatty replacement, we hypothesized that functional conduction block could cause the ST-segment elevation. The homogeneous region of high resistance was replaced by a region of inhomogeneous tissue, using random variations at a resolution of 0.25 mm as an approximation to the myopathic ventricle. This adaptation caused the same delays as in homogeneous tissue with increased resistance albeit at reduced amplitude of the local deflection. Reduction of the sodium current to 30% of its normal value led to further activation delay in RV/RVOT, followed by conduction block due to source-sink mismatch where the activation front reached an area of lower resistance. Local deflections in the electrogram were small and delayed, or absent (at more distal sites). In the ECG, coved-type ST-segment elevation and negative T-waves were now observed in right precordial leads. The model has also been used to estimate the relative contributions of various parts of the heart to the surface ECG, both at baseline and during ajmaline infusion. Results show that in none of the precordial leads is the influence of the right ventricle larger than from the left. Contribution of the RVOT to the ECG is small, but largest in V1 and V2. Status: The study is ongoing; detailed histology of the right ventricle (and its outflow tract) will be performed, BSM data of Brugada patients will be analyzed and used for electrocardiographic imaging (determining epi and endocardial activation from body surface recordings, structural abnormalities in the right ventricle and its outflow tract will be incorporated in the computer model and the overlap syndrome will be simulated with the model. Annual Report 2007

20 20 Investigators: AC Linnenbank (ICIN, NHS), M Potse (ICIN, NHS), PG Postema, (UvA), PFHM van Dessel, (UvA), LRC Dekker (UvA), AAM Wilde, (UvA), P Loh (UMCU), HL Tan (UvA), M Hoogendijk (UvA), R Coronel (UvA), JMT de Bakker, (ICIN). Financial support: This study is granted by the Dutch Heart Foundation (2005B092). Enhanced susceptibility to ventricular arrhythmias in human and murine heart failure is associated with heterogeneous distribution of connexin43 Background: Structural and electrical remodeling during congestive heart failure (CHF) in patients is accompanied by high susceptibility to life-threatening ventricular tachyarrhythmias (VT). To investigate the underlying arrhythmogenic mechanism, we studied cardiac remodeling and arrhythmogenicity in CHF patients and in a mouse model of chronic pressure overload. Methods and results: From end-stage CHF patients with (VT+, n=7) or without (VT-, n=8) a history of VT, myocardial tissue biopsies were available. Biopsies from donor hearts rejected for transplantation served as control (n=10). Mice with transverse aortic constriction (TAC, n=18) were compared to sham operated mice (n=19) and were subjected to Doppler echo and electrocardiography 16 weeks after operation. Epicardial activation mapping was performed on LV and RV of isolated hearts. Arrhythmia susceptibility was determined by programmed stimulation. Tissue was processed for Connexin43 (Cx43) immunohistochemistry and Sirius Red staining of collagen. In CHF patients, ejection fraction (EF) was decreased (17±1%) and QRS duration was increased (131±3 ms) compared to controls. Neither EF nor QRS duration was different between VT+ and VT- patients. In TAC mice, fractional shortening (FS) was decreased (38.4±2.3 vs sham 46.3±1.9%; p<0.5), QRS duration was increased (12.4±0.4 vs sham 9.5±0.4 ms; p<0.01), and RV epicardial longitudinal conduction velocity was reduced (39.0±1.8 vs sham 50.9±2.7 cm/s; p<0.5). Polymorphic VTs HUMAN MOUSE were induced in 8/18 TAC and 0/19 sham mice (p=0.01) and were based on reentry. Comparable to CHF patients, QRS duration and FS were similar in TAC mice with and without arrhythmias. Interstitial fibrosis was significantly higher in both CHF patients and TAC mice compared to controls, but not different between VT+ and VT-. Interestingly, heterogeneously distributed Cx43 expression was exclusively found in VT+ patients and TAC mice with arrhythmias, while Cx43 expression was normal in VT- patients and TAC mice without arrhythmias (Figure 2). Heterogeneous redistribution of Cx43 protein in CHF patients and TAC mice is associated with increased arrhythmia vulnerability. Status: This project has been concluded and a manuscript is ready for submission Investigators: M Boulaksil (ICIN, NHS), SKG Winckels (UMCU), MA Engelen (UMCU), M Stein (UMCU), AAB van Veen (ICIN, NWO), JA Jansen (UMCU), AC Linnenbank (ICIN, NHS), MFA Bierhuizen (UMCU), MFM van Oosterhout (UMCU), H Kirkels (UMCU), N de Jonge (UMCU), A Varró (University of Szeged, Szeged, Hungary), MA Vos (UMCU), JMT de Bakker (ICIN), HVM van Rijen (UMCU). Financial support: This study is granted by the Netherlands Heart Foundation (M96.001). Tissue engineering: Mechanisms of differentiation in human fetal cardiac progenitor cells Background: In collaboration with the Department of Cardiology of the UMC Utrecht (Marie-José Goumans and Pieter Doevendans) we previously demonstrated that human cardiac progenitor cells (CPCs) can be differentiated efficiently into rather mature cardiomyocytes. The exact mechanisms triggering differentiation in vitro or in vivo are not clear yet, therefore we investigated whether hyperpolarization of CPCs contributes to differentiation. Methods and results: CPCs were cultured in media with low potassium concentration (LowK) and allowed to differentiate for several weeks. Subsequently, expression of cardiac markers was determined with RT-PCR and electrophysiological and calcium-imaging approaches were used to determine the effects of hyperpolarization on membrane potential and intracellular calcium concentration. Additionally, CPCs were transfected with an NFAT luciferase construct to monitor activation of the calcineurin pathway. Lowering the potassium concentration resulted in a hyperpolarization of the undifferentiated CPCs from approximately -30 mv to -40 mv. Calcium imaging experiments showed a significantly increased calcium concentration after 4 hours of LowK medium. Results from the NFAT luciferase assays demonstrated enhanced NFAT activation. After several weeks, LowK treated cultures displayed up-regulated expression of cardiac markers such as βmhc and α-actinin. Resting membrane potential of LowK differentiated cultures was approximately -60 mv. Figure 2: Typical examples of anti-connexin43 labeling in CHF patients (Panel A) and in mice (Panel B). Human examples of LV anti-cx43 labeling are shown for heart donors (Ctrl) and CHF patients with or without a history of ventricular tachycardia (VT+/VT-). Cx43 expression is not different for VT+ and VT- neither in man nor mice. Quantifications of heterogeneity of Cx43 distribution are shown in panel B. Status and continuation: This study is almost completed and a manuscript is in preparation. Investigators: TP de Boer (ICIN, UMCU), P van Vliet (UMCU), JPG Sluiter (UMCU), JMT de Bakker (ICIN), PA Doevendans (UMCU), AAB van Veen (NWO, ICIN), MJ Goumans (NWO), SCM van Amersfoorth (AMC), MAG van der Heyden (STW, ICIN) Interuniversity Cardiology Institute of the Netherlands

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