days of molecular medicine
The meeting was organised by the UCSD Institute of Molecular Medicine and the nearby Salk Institute in collaboration with Nature Medicine. The title of the meeting was heart and brain: signalling pathways in complex human diseases. For the readers of this journal, the focus of this report is on the heart.
Scientiflc session I Calcium signals and disease pathways Calcium-MEF-2 signalling in cardiac disease was discussed by Eric Olson (Dallas, US). His seminal work on the unravelling of cardiac hypertrophy is unique in the field. He focussed on the downstream pathway of calmodulin by looking at the steps involved in activation ofCAM kinase all the way to the transcription factor MEF2c. Skeletal muscle differentiation is controlled by interactions between myocyte enhancer factor-2 (MEF2) and myogenic basic helix-loop-helix transcription factors. Association of MEF2 with histone deacetylases (HDAC) 4 and 5 results in repression of MEF2 target genes and inhibition of myogenesis. Calcium/ calmodulin-dependent protein kinase (CaMK) signalling promotes myogenesis by disrupting MEF2HDAC complexes and stimulating HDAC nuclear export. To further define the mechanisms that confer CaMK responsiveness to HDAC4 and 5, this group performed yeast
two-hybrid screens to identify HDAC-interacting factors. These screens revealed interactions between
HDAC4 and members of the 14- A selected oral presentation was 3-3 family of proteins, which given by Aylin Rodan. She discussed function as signal-dependent intra- the effects ofethanol on drosophila cellular chaperones. HDAC4 binds and the role of various brain areas constitutively to 14-3-3 protein in involved in the response to ethanol. yeast and mammalian cells, whereas She introduced a fantastic device HDAC5 binding to 14-3-3 is large- called the 'inebriometer', basically a ly dependent on CaMK signalling. series of vertically connected glass CaMKphosphorylates serines -259 compartments with ethanol at the and -498 in HDAC5 subsequently bottom. Her experiments with flies serving as docking sites for 14-3-3. showed that when they are drunk These studies suggest that 14-3-3 they lose postural control and fall to binding to HDAC5 is required for the bottom. The mean elution time CaMK-dependent disruption of of a fly population can be used as a MEF2-HDAC complexes and nu- measure of sensitivity to ethanol. She clear export of HDAC5, and im- showed increased sensitivity in flies plicate 14-3-3 as a signal-dependent with a mutation in adenyl cyclase. regulator of muscle cell different- By expressing a protein kinase Ainhibitory protein in different iation. compartments of the brain, the The role of CAM kinases in neuronal dominant region for alcohol signalling and disease was high- sensitivity was identified to be a very lighted by Dr Howard Schulman small subset of central complex (San Francisco, US). In itself CAM neurons. kinase is a very interesting protein. The protein is built from 12 re- Scientific session 11, Thursday gulatory subunits organised as two 14 March hexamers. The speaker showed beautiful work The diseases of excitability. on the dynamics of CAM kinase A fascinating new field in ion activity. CAM kinase is a protein channel studies was discussed by subjected to autophosphorylation David Clapham (Boston, US). He and the activation of the protein introduced a completely new class of depends on the stimulus frequency, ion channels. TRP channel proteins which is important in tissues like constitute a large and diverse family brain and heart. It is a mode for the of proteins that are expressed in cell to adapt calcium control to many tissues and cell types. This changes in the firing rate. CAM family was designated TRP because kinase interacts with ankyrin pro- of a spontaneously occurring teins, NR2B unit of the NMDA drosophila mutant lacking TRP that receptor and the L-type calcium responded to a continuous light channel. Activation ofCAM kinase with a transient receptor potential could contribute to a pro- (hence TRP). In addition to arrhythmogenic state developing responses to light, TRPs mediate responses to nerve growth factor, early after depolarisations.
Netherlands Heart Joumal, Volume 10, Number 9, September 2002
pheromones, olfaction, mechanical and chemical factors, temperature, pH, osmolarity, vasorelaxation of blood vessels and metabolic stress. More then 20 proteins have been identified thus far. These different proteins are divided into three classes: short, vanilloid and long. Clapham addressed in detail the TRP-PLIK channel, and one ofthe reasons for working on this isoform is the unique combination of a channel protein that also has a kinase domain. The channel subunits have six transmembrane domains that most probably assemble into tetramers to form non-selective cationic channels, which allow for the influx of calcium ions into cells. The kinase segment dimerises and resembles protein kinase A. The TRP-PLIK activating pathways were analysed and activation ofthe protein appeared to be independent of Ca2+ buffers, diacylglycerol, and inositol trisphosphate formation. Activity of the channel is modified by PIP2 and Gq receptor proteins. In the brain these channels are important for pain perception.
The interaction between Yotaio and KCNQ1 was mediated by a leucine zipper domain. In one Finnish family the LQTS1 causing mutation was found in this domain, preventing the docking of the protein complex and causing an abnormal response to adrenergic stimuli.
plete absence of transient outward potassium current, It,, and a marked increase in action potential duration. Transcription factors, cytoskeletal and ion channel genes are all essential for a normal cardiac morphology and contractile and electrical function. The mouse models created provide insight in the mechanisms of sudden cardiac death. The knowledge obtained from these mouse studies will help us to understand and possibly prevent this dramatic event in human.
The next speaker was the host ofthe meeting, DrKen Chien (San Diego, US). He stressed the high incidence of sudden death and the resulting major impact on our society. Although several genetic causes have been unravelled, in the majority of patients the cause and mechanism Scientific session III remain to be established. By developing mouse models more Plasticity, differentiation and insight has been obtained in diseases disease such as right ventricular dysplasia, It is always a joy to listen and look proarrhythmic defects in the to the work of Mark Fishman conduction system (HF-lb) and (Boston, US). His topic 'genetics of changes in channel gene expression the first heart beat in zebrafish' is in cardiac failure. always illustrated with impressive The model for right ventricular registrations ofcardiac abnormalities dysplasia was created by disruption in the fish heart. The approach is of the gene encoding the cyto- based on a mutagenesis screen in skeletal protein ALP. ALP-deficient zebrafish to identify cardiac phenomice develop RV chamber dilation types. This time he discussed and dysfunction, directly implicating mutant lines with impaired growth A superb teaching lecture was given a-actinin-associated proteins in the indicated by 'Heart of glass, Island by Dr Robert Kass (New York, US). onset of cardiomyopathy. In vitro beat and Liebeskummer (named by He introduced the basics ofthe long assays showed that ALP directly a German postdoc in the lab). QT syndrome. There is a marked enhances the capacity of a-actinin Heart of glass indicates the lack of difference in the triggers related to to cross-link actin filaments, indi- concentric growth, which leaves the arrhythmic events in families with cating that the loss ofALP function ventricle almost transparent. In mutations in different genes. Mu- contributes to destabilisation of island beat a reduction of the tations in KCNQ1 (LQTS1) lead to actin anchorage sites in cardiac number of ventricular cells was sudden death upon sympathetic muscle. ALP also colocalises at the observed which lead to impaired stimulation such as exercise. Despite intercalated disc with a-actinin and cardiac function. By positional knowledge on the exact location of y-catenin, the latter being a known cloning the mutation was found in the mutation, the role of the sym- disease gene for human RV dys- the al C L-type calcium channel subunit (C-LTCC). The atrium is pathetic nervous system is not yet fully plasia. understood. His group showed the KChIP2, a gene encoding three relatively normal in size, and indiimportance of looking at the protein auxiliary subunits of Kv4.2 and vidual cells contract chaotically, in a complex rather then the isolated ion Kv4.3, is preferentially expressed in pattern resembling atrial fibrillation. channel. On the intracellular domain, the adult heart, and its expression is The ventricle is completely silent. several proteins attach including downregulated in cardiac hyper- The phenotype of the Liebeskumprotein phosphatase 1, protein kinase trophy and failure. Mice deficient mer fish is characterised by hyperA, and a kind of docking protein for KChIP2 exhibit normal cardiac trophy and an increase in the cell labelled Yotaio. This complex of structure and function but display a numbers (hyperplasia) The phenofactors co-immunoprecipitates with prolonged elevation in the ST type is related to mutations in reptin the L2 domain of KCNQ1. The segment on the electrocardiogram. (repressor of pontin). Reptin and presence of a kinase and phosphatase The KChIP2 deficient mice are pontin are transcription factors allows the complex to respond to highly susceptible to the induction involved in [ catenin activation. The adrenergic stimuli, which was further of cardiac arrhythmias. Single-cell expression ratio between reptin and documented in electrophysiological analysis revealed a substrate for pontin determines growth. Reptin studies. arrhythmogenesis, including a com- blocks and pontin stimulates 388
Netherlands Heart Joumal, Volume 10, Number 9, September 2002
growth. Some zebrafish models resemble human disease, such as Holt Oram syndrome (TBX5 mutation) and dilated cardiomyopathy (Titin gene).
completely. Through a mutation screen, genes were uncovered that are essential for this process. One of the genes was named nightcap, based on the abnormal fin morphology after regrowth. The mutated gene turned out to be MPS1 protein kinase, an essential gene in the cell cycle and involved in centrosome duplication and the spindle assembly checkpoint in mouse and yeast. Another interesting gene is MSX1, a gene involved in dedifferentiation, an essential mechanism preceding tissue regeneration. Another fascinating zebrafish characteristic is the complete recovery of the ventricle after dissection ofthe apex. Not only do myocytes start to divide again, the regeneration encompasses all cell types and also the myocardial architecture. Again zebrafish with mutated MSX1 and MPS-1 genes revealed impaired regenerative capacity.
Cytoskeletal pathways for cardiac and muscle disease was the topic of the presentation by Mary Beckerle (Utah, US). Her work is based on the identification of proteins involved in the anchorage of actin to the cytoskeleton. One of the key players here is the cx-actinin gene. The speaker used protein overlay (based on immunoprecipitation) to identify proteins interacting with aactinin. The proteins involved included Cysteine Rich Protein (CRP) and a-actinin associated LIM protein or ALP. One of the CRP isoforms (3) is the Muscle Lim Protein (MLP). The MLP-deficient mouse is a well-established model for inherited dilated cardiomyopathy. A mutant MLP protein in drosophila leads to flight defects. HIF- 1: oxygen homeostasis and MLP is important for anchorage of disease pathology was the title ofthe the cytoskeleton to the outer mem- presentation by Gregg Semenza brane through spectrin binding and (Baltimore, US). Hypoxia-inducible to the sarcomere by binding the T factor 1 (HIF- 1) activates transcap of titin. In addition, the protein cription of genes encoding proteins contributes to gene expression that mediate adaptive responses to regulation in skeletal muscle by reduced oxygen availability. The interaction with the transcription HIF-1j subunit is constitutively factor MyoD. The ALP protein expressed, whereas the HIF-la improves binding of a-actinin to subunit is subject to ubiquitination actin. In addition, an interaction of and proteasomal degradation, a ALP with transcription factor process that is inhibited under myogenin has been documented in hypoxic conditions. Recent data skeletal muscle. ALP deficiency in indicate that HIF- 1 plays major mice leads to right ventricular car- roles in the prevention of myocardial diomyopathy; in C. Elegans im- and cerebral ischaemia and in the paired contractility was documented. pathogenesis of pulmonary hypertension and cancer. One of the Session IV important effects of HIFI expression is the induction of VEGF Environmental stress and cell expression. Modulation of HIF-1 survival pathways activity by genetic or pharmaMark Keating (Boston, US), who cological means could provide a obtained his fame in human novel therapeutic approach to these genetics, surprised the audience common causes of mortality. with a complete switch to the zebra- Hypoxia-inducible factor 1 (HIFfish as a model for genetic analysis. 1) is a master regulator of oxygen The key question was on tissue homeostasis that controls angioregeneration. The zebrafish has a genesis, erythropoiesis, and glyremarkable regenerative capacity. If colysis via transcriptional activation a fin is removed it will be replaced of target genes under hypoxic Netherlands Heart Joumal, Volume 10, Number 9, September 2002
conditions. 02-dependent binding of the von Hippel-Lindau (VHL) tumour suppressor protein targets the HIF-la subunit for ubiquitination and proteasomal degradation. The activity of the HIF-la transactivation domains is also 02 regulated. A new protein, Factor Inhibiting HIF-1 (FIH-1), has been identified that binds to HIF-la and inhibits its transactivation function. In addition, FIH-1 binds to VHL and VHL also functions as a transcriptional co-repressor that inhibits HIF-la transactivation function by recruiting histone deacetylases. Involvement of VHL in association with FIH-1 provides a unifying mechanism for the modulation of HIF-la protein stabilisation and transcriptional activation in response to changes m cellular 02 concentration. A serious attempt to integrate brain and heart research was undertaken by Carmen Birchmeier (Berlin, Germany). The signalling system comprising the ligand neuregulin1 and its receptors, ErbB2 and ErbB3, plays multiple and important roles in glial development. The ErbB2 tyrosine kinase functions as co-receptor for the neuregulin receptors ErbB3 and ErbB4 and can participate in signalling of the EGF receptor (ErbBI ), interleukin receptor gpl30, and G-protein coupled receptors. Amplification of the gene encoding the ErbB2 (Her2/neu) receptor tyrosine kinase is critical for the progression ofseveral forms ofbreast cancer. In a large-scale clinical trial, treatment with Herceptin (trastuzumab), a humanised blocking antibody against ErbB2, led to marked improvement in survival. However, cardiomyopathy was uncovered as a mitigating side effect, thereby suggesting an important role for ErbB2 signalling as a modifier of human heart failure. ErbB2(-/-) mice die at midgestation because of heart malformation. A genetic rescue ofheart development by myocardial expression of erbB2 cDNA has been implemented, which allows survival of the mutants to birth. In rescued
erbB2 mutants, Schwann cells are lacking. Motoneurons form and can project to muscle, but nerves are poorly fasciculated and disorganised. In addition, a severe loss of motoneurons at the cervical and lumbar but not at thoracic levels occurs. The results define the roles of Schwann cells during motoneuron and synapse development, and reveal different survival requirements for distinct motoneuron populations. To investigate the physiological role of ErbB2 signalling in the adult heart, mice with a ventricular-restricted deletion ofErbb2 were generated and studied. These ErbB2-deficient conditional mutant mice were viable and displayed no overt phenotype.
However, physiological analysis revealed the onset of multiple independent parameters of dilated cardiomyopathy, including chamber dilation, wall tiinning and decreased contractility. ErbB2 signalling in cardiomyocytes is therefore essential for the prevention of dilated cardiomyopathy, and reveals different survival requirements for distinct motoneuron populations. Epidemiology has developed as a powerfiul tool for determining the genetic basis for predisposition to certain diseases. Dr Ming Tsuang presented data associating particular polymorphisms with a predisposition for schizophrenia, all ofwhich had
been identified in families where schizophrenia presented in several family members. In addition to the polymorphisms, he showed data for a locus at chromosome 15qI4; however the responsible gene remains to be determined. He revealed that treatment with low doses ofstandard schizophrenia drugs in phases where only mild symptoms of the disease had presented (e.g. asocial behaviour) was sufficient to delay/prevent its full development in patients with genetic susceptibility. U
P.A. Doevendans. C. Mummery. HubrechtIaboratory Utrecht, ICIN
WCN Research Congres Aspecten van klinisch onderzoek in de cardiologie Ermelo, 8 november 2002 De WCN (Working group on Cardiovascular research in the Netherlands) bestaat 15 jaar. In de huidige vorm van vereniging (sinds december 1999) is de WCN uitgegroeid tot een professionele organisatie van 62 perifere cardiologische klinieken. Naast de WCN cardiologen, die zich met klinisch onderzoek bezig houden, hebben deze klinieken meer dan 150 medewerkers in dienst die het klinisch wetenschappelijk onderzoek uitvoeren.
De WCN hecht veel waarde aan bevorderen en bewaken van kwaliteit van onderzoek in haar klinieken. Kwaliteit wordt voor een groot deel bepaald op de werkvloer. Vanafheden zal jaarlijks een separaat 'Research Congres' worden georganiseerd ter vervanging van de parallelsessie voor research medewerkers tijdens het WCN december congres. Bij dit Research Congres staan de aspecten van klinisch onderzoek centraal. Dit congres is bedoeld
voor research artsen, coordinatoren, verpleegkundigen, cardiologen, assistenten en medewerkers, leden VMPO, medewerkers CRO's en farmaceutische industrie. Het congres zal in het Nederlands worden gehouden. Het belooft een interessante en afwisselende dag te worden. U bent van harte welkom. Marion van der Heijden, managing director WCN, bestuur WCN
Ochtendprogramma o.I.v. drs. Herman Pieterse 10.00 uur Wet- en regelgeving nationaal/ intemationaal 'Hoe staat Nederland ervoor na 2 jaar WMO; lopen we voor of achter op de intemationale afspraken?' 11.30 uur Good Clinical Practice (GCP). 'De nieuwe Europese richtlijnen; wat zijn de consequenties 12.30 uur Lunchpauze en bezoek stands/ exhibitie Middamma 13.30 uur Programma met keuze wit de volgende workshops: a. Projectmanagement - algemeen en in een reeds bestaande structuur b. Informed Consent Procedure - achtergronden, procedure, valkuilen c. Laboratorium - achtergronden en praktische oefening; de praktijk 15.45 uur Plenaire discussie a.d.h.v. actuele onderwerpen ingebracht door deskundige sprekers. 17.30 uur Einde van het dagprogramma en aanvang informeel gedeelte 19.00 uur Diner en feest
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Netherlands Heart Journal, Volume 10, Number 9, September 2002