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ObjectivesThis study sought to explore whether subclinical alterations of sarcoplasmic reticulum (SR) Ca2+ release through cardiac ryanodine receptors (RyR2) aggravate cardiac remodeling in mice carrying a human RyR2R4496C+/– gain-of-function mutation in response to pressure overload.BackgroundRyR2 dysfunction causes increased diastolic SR Ca2+ release associated with arrhythmias and contractile dysfunction in inherited and acquired cardiac diseases, such as catecholaminergic polymorphic ventricular tachycardia and heart failure (HF).MethodsFunctional and structural properties of wild-type and catecholaminergic polymorphic ventricular tachycardia–associated RyR2R4496C+/– hearts were characterized under conditions of pressure overload induced by transverse aortic constriction (TAC).ResultsWild-type and RyR2R4496C+/– hearts had comparable structural and functional properties at baseline. After TAC, RyR2R4496C+/– hearts responded with eccentric hypertrophy, substantial fibrosis, ventricular dilation, and reduced fractional shortening, ultimately resulting in overt HF. RyR2R4496C+/–-TAC cardiomyocytes showed increased incidence of spontaneous SR Ca2+ release events, reduced Ca2+ transient peak amplitude, and SR Ca2+ content as well as reduced SR Ca2+-ATPase 2a and increased Na+/Ca2+-exchanger protein expression. HF phenotype in RyR2R4496C+/–-TAC mice was associated with increased mortality due to pump failure but not tachyarrhythmic events. RyR2-stabilizer K201 markedly reduced Ca2+ spark frequency in RyR2R4496C+/–-TAC cardiomyocytes. Mini-osmotic pump infusion of K201 prevented deleterious remodeling and improved survival in RyR2R4496C+/–-TAC mice.ConclusionsThe combination of subclinical congenital alteration of SR Ca2+ release and pressure overload promoted eccentric remodeling and HF death in RyR2R4496C+/– mice, and pharmacological RyR2 stabilization prevented this deleterious interaction. These findings suggest potential clinical relevance for patients with acquired or inherited gain-of-function of RyR2-mediated SR Ca2+ release.

Triggered activity induced by delayed afterdepolarizations has been investigated extensively in tissues isolated from different sites in the heart and from different species. In spite of this we still do not know the clinical relevance of this arrhythmogenic mechanism. An answer to this question is important because the ability to identify mechanisms in the clinical situation can improve our understanding of pathophysiology and provide new treatments. Presently, different methods are used to discriminate among mechanisms of arrhythmias, including electrical stimulation [1], antiarrhythmic drugs, and recording of monophasic action potentials. Unfortunately no method has proven to be sufficiently sensitive or specific [2, 3].

Diastolic Ca2+ leak from the sarcoplasmic reticulum (SR) via the ryanodine receptor (RyR2) contributes to arrhythmias. Protein kinase A (PKA) and Ca2+/Calmodulin- dependent protein kinase II (CaMKII) have been involved in SR Ca2+ leak by altering RyR2 gating. Under these conditions, JTV519 (K201), a benzothiazepine derivate, has been shown to stabilize the modified RyR2 and reduce diastolic SR Ca2+ leak. We tested, whether JTV519 reduces SR Ca2+ leak induced by Na+- and Ca2+ overload (induced by 100 µM ouabain), i.e. independent of CaMKII signaling. Methods: [Ca2+]i was measured (Fluo4-AM, confocal) in paced murine cardiomyocytes ±JTV519 (1 µM, >1h preincubation). [Ca2+]-transients, diastolic SR Ca2+ leak (Ca2+ spark (SparkF, in s−1∗pL−1) and Ca2+ wave frequency) and SR [Ca2+] (caffein) were measured ±ouabain (OUAB) and KN93 (CaMKII-inhibitor, 1 µM). Phosphorylation of RyR2 (pSer2814, CaMKII site) was quantified by Western blot.Results: With OUAB, total cellular [Ca2+] increased from 3.3±0.3 to 4.3±0.3 (F/F0, mean±SE), SparkF increased from 31±20 to 85±30 (both pl0.05). Ouabain did not increase pSer2814, and KN93 had no effect on elevated SparkF with ouabain (89±24), indicating no contribution of CaMKII to increased SR Ca2+ leak. JTV519 decreased SparkF (25±4), and Ca2+ waves, but also attenuated the increase in SR [Ca2+] in OUAB (F/F0: 5.2±0.3 with JTV+Ouab vs. 7.7±0.6 OUAB, pl0.05). However, matching cells for equal SR [Ca2+] revealed an SR [Ca2+] - independent effect of JTV519 on SparkF. In contrast, propagation speed of Ca2+ waves and the ratio between [Ca2+] transient amplitude and SR [Ca2+] were unchanged.Conclusion: In conditions not related to CaMKII-mediated alterations of RyR2 gating, JTV519 reduces spontaneous SR Ca2+ release, but does not influence propagated Ca2+ release or the fraction of SR Ca2+ released with each beat.

Pharmacological safety evaluation of (pro) drugs includes cardiac safety assessment of proarrhythmic liability in healthy tissue with emphasis on the rapid component of the delayed rectifier (I(Kr)). The lack of (1) an arrhythmic end point, (2) tests in remodeled, predisposed tissue, and (3) testing chronic drug influence on channel trafficking impairs on the drawn conclusions of these assays regarding drug safety. Moreover, the currently used human ether-à-go-go-related gene assays, action potential duration, prolongation in multicellular preparations, or the QT interval have significant shortcomings in their prediction of an increased risk for drug-induced torsades de pointes arrhythmia. In this review, it will be proposed that beat-to-beat variability of repolarization quantified as short-term variability can (1) discriminate between safe and unsafe drugs even under predisposed and highly arrhythmogenic conditions despite accompanying QT prolongation and (2) identify the individual at risk for subsequent arrhythmic events.

Promoting therapy adherence requires understanding various psychosocial parameters, including patients’ need for information. Drug information adapted to patients’ needs may empower them and increase their confidence in drug therapy. Objectives: To explore psychiatric in-patients’ information preferences and to test the reliability of a Dutch version of the Intrinsic Desire for Information (IDI) scale in psychiatric institutions in Flanders. Standardised interviews were conducted with psychiatric patients in 11 hospitals. The interview consisted of the IDI-scale and five open questions. Patient demographics collected were sex, age, number of medicines taken, diagnosis, number of admissions during the past year, marital status, education level and occupation. 279 patient interviews were completed. A factor analysis on the original 12-item scale yielded 3 factors. An abbreviated scale was derived from the first factor (F1). This 6-item scale measured ‘extent of information desired:’ (EID) and consisted of six items (Cronbach’s α = 0,73). A second factor (F2) measured ‘information provider preference’ (IPP) (α = 0,56) and a third factor (F3) measured ‘inhibited information desire’ (IID) (α = 0,69). EID was associated with number of medicines taken, duration of hospitalisation and marital status. The internal reliability of the EID-factor appears to be reproducible in the specific setting of psychiatric hospitals. It may be useful to help healthcare professionals develop pharmaceutical care towards psychiatric patients. Validation of the scale remains to be completed. Information need in psychiatric in-patients measured by the EID-score was comparable to the need measured in general hospitals during earlier research in England. Targeted information services seem to be desirable to enhance therapy adherence and quality of life in psychiatric patients.

The novel antiarrhythmic drug K201 (4-[3-{1-(4-benzyl)piperidinyl}propionyl]-7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine monohydrochloride) is currently in development for treatment of atrial fibrillation. K201 not only controls intracellular calcium release by the ryanodine receptors, but also possesses a ventricular action that might predispose to torsade de pointes arrhythmias. The anti- and proarrhythmic effects of K201 were investigated in the anesthetized canine chronic atrioventricular block model. Two doses of K201 (0.1 and 0.3mg/kg/2 min followed by 0.01 and 0.03 mg/kg/30 min i.v.) were tested in 4 serial experiments in dogs with normally conducted sinus rhythm (n=10) and in torsade de pointes-susceptible dogs with chronic atrioventricular block. Susceptibility was assessed with dofetilide (0.025 mg/kg/5 min i.v.). Beat-to-beat variability of repolarization was quantified as short-term variability of left ventricular monophasic action potential duration. In dogs with normally conducted sinus rhythm, both doses of K201 prolonged ventricular repolarization whereas only the higher dose prolonged atrial repolarization. At chronic atrioventricular block, dofetilide induced torsade de pointes in 9 of 10 dogs. K201 did neither suppress nor prevent dofetilide-induced torsade de pointes. K201 dose-dependently prolonged ventricular repolarization. In contrary to the lower dose, the higher dose did increase beat-to-beat variability of repolarization (from 1.2 ± 0.3 to 2.9 ± 0.8 ms, P

Myocardial strain imaging could potentially improve patient selection for cardiac resynchronization therapy (CRT). Available techniques are CMR tagging (CMR-TAG), feature tracking (CMR-FT) and speckle tracking echocardiography (STE). Despite promising results per modality, a direct comparison is

In this context, the CLEVER project has the intention to analyse and answer these different questions. The objectives of the project can be described as follows, with a focus on the passenger car market: * Create an objective image of the environmental impact of vehicles with conventional and alternative fuels and/or drive trains; * Investigate which price instruments and other policy measures are possible to realise a sustainable vehicle choice; * Examine the external costs and verify which barriers exist for the introduction of clean vehicle technologies on the Belgian market; * Analyse the global environmental performances of the Belgian car fleet; * Formulate recommendations for the Belgian government to stimulate the purchase and use of clean vehicles. To achieve these objectives, a multidisciplinary approach has been used, in which the different tasks are performed by the different partners. On the basis of a literature review, a preliminary "state-of-the-art" has been carried out on different topics, more specifically on vehicle technologies, existing environmental vehicle assessments, policy measures and consumer behaviour for the purchase of cars. To compare the environmental impacts of vehicles with different conventional (diesel, petrol) and alternative fuels (LPG, CNG, alcohols, biofuels, biogas, hydrogen) and/or drive trains (internal combustion engines and battery, hybrid and fuel cell electric vehicles), a Life Cycle Assessment (LCA) is performed, within a Belgian context. LCA studies the environmental aspects and potential impacts of a product throughout its life from raw material acquisition through production, use and disposal and presents the advantage of being standardized (ISO 14040 & 14062, 2006). Next to the well-to-wheel emissions (related to fuel production, transportation and fuel use in the vehicle), which is assessed in the Ecoscore methodology, the LCA also includes cradle-to-grave emissions (related directly and indirectly to vehicle production and end-of-life processing of the vehicle). The final aim is to develop a methodology with a per-model applicability. A detailed description of the different tasks of the LCA approach (software selection, inventory and data collection, classification and characterisation, sensitivity and probability analysis, scientific validation of the Ecoscore approach) is described further in chapter 2. To compare the cost-efficiency of different vehicle technologies, the Life Cycle Cost (LCC) methodology has been used. From a user perspective, the LCC is often a crucial factor. Life cycle costs are all the anticipated costs associated with a car throughout its life and include all user expenses to own and use vehicles. The LCC consists of the vehicle financial costs (purchase price, governmental support, registration tax), fuel operational costs and non fuel operational costs (yearly taxation, insurance, technical control, battery, tyres and maintenance). The used method within the LCC analysis is the net present value method as one has to accurately combine the initial expenses related to the purchase of the car with the future expenses related to the use of the car. A further description of the methodology and results of this task are described in chapter 3. The proposed policy measures will only be effective if they induce the right behavioural responses. That is why in a first phase a literature review of price elasticities has been performed. Additionally, price sensitivities are empirically derived through the development of a "green vehicle demand model", which enables to estimate the distribution of respondents willing to switch to a more environmentally friendly car, based on different weighted pricing levels of combined policy measures (chapter 4). The different tasks are supported with inputs of state-of-the-art external cost factors. The "ExternE" methodology for the calculation of external costs of transportation is updated and adapted for its use in a Belgian context. Attention has been paid to the best methods and their updating, in order to quantify the external effects associated with new vehicle technologies. Thanks to the knowledge of the externalities, the environmental cost can be integrated into the life cycle cost analysis of new vehicles. This approach allows a complete comparison with conventional vehicles, based on a full-cost approach (chapter 5). The main barriers impeding the development of alternative vehicles (with alternative fuels and propulsion systems) in Belgium as well as their relative importance have been identified. This objective is approached through the consultation of the different groups of stakeholders. Barriers can be grouped into the following categories: economic, technical, psychological, legislative, political, institutional, environmental/societal, market, supply and demand barriers. Strong relationships exist between the different barriers; in fact, they are integrated into an aggregation of complex causal connections. The second original objective is to derive a systemic scheme representing the inter-relations between barriers. This allows for a more global view on the barriers, which is essential for drawing effective policy measures (chapter 6). Price instruments are suitable to integrate the environmental performance of vehicles in this purchase decision. The CLEVER project allows investigating possible policies towards a more sustainable car choice (chapter 7). Implementation pathways for a consistent policy for the promotion of cleaner vehicles are being developed. These possible policies are price policies (road pricing, fiscal measures, modulated vehicle taxation, parking prices, subsidies...), regulatory policy, etc. The investigated policy instruments not only focus on individual vehicle-buying behaviour but also on policies towards companies and public authorities. The pathways have been developed based on the analysis of the environmental impact, the barriers for the purchase and use of cleaner vehicles. This was done in parallel with the international review of policy measures and related research and consultation of the different target groups in Belgium. The road emission model from VITO has been used to assess the global environmental performance of the whole Belgian vehicle fleet. From this model the Ecoscore module is applied to the different vehicle categories (defined by fuel, age, engine size, etc.) of the road emission model to result in a combined Ecoscore-emissions-road model. This allows generating an indicator of the global environmental performance of the fleet and making projections on how this will evolve in time in different scenarios. The projections have been done for the years 2010 up to 2030 in steps of 5 years, with the mid-term timeframe being 2020 and the long-term timeframe being 2030. Three scenarios have been calculated within these timeframes, and one additional, more visionary, scenario has been calculated solely for 2060. Finally, by means of a multi-criteria analysis (MCA), the scenarios (baseline, realistic, progressive), elaborated in the previous chapters, have been evaluated on several criteria for which input has been gathered throughout the other tasks of the CLEVER project (chapter 8). For this purpose, a combination of the PROMETHEE methodology and the Analytic Hierarchy Process (AHP) is used. The overall aim is not to categorize the single best scenario, but to formulate suitable policy recommendations to the decision makers which take into consideration the weak and the strong points of the considered scenarios. To finalise this project, the main conclusions of all the different tasks, gathered by the different partners, have been combined in the last section of this report (chapter 9). In this chapter, answers are formulated on the different research questions of the project, which are the basis of recommendations for policy makers on how to stimulate the purchase and use of clean vehicles in a Belgian context.