Financiación de Proyectos de Investigación Traslacional. Documento A: Solicitud Proyecto. I. Datos del Proyecto Número de Registro:

(1)

Financiación de Proyectos de Investigación Traslacional

Documento A: Solicitud Proyecto

I. Datos del Proyecto Número de Registro:

Título del Proyecto

Remodeling of adenosine receptors in human atrial fibrillation: A novel mechanism for atrial arrhythmogenesis

Duración 3 años X 4 años 5 años

Número de Sub-proyectos 4

II. Datos del Centro Coordinador Denominación

Hospital de la Santa Creu i Sant Pau

Domicilio

Sant Antoni Mª Claret, 167 Ciudad

Barcelona

Provincia Barcelona

Código Postal 08025

Dependencia

Servicio de Cardiología

Teléfono 93.556.59.45 III. Datos del Representante Legal del Centro Coordinador Nombre

Jordi

1º apellido Colomer

2º apellido Mascaró Denominación del Cargo

Director General

Dirección de contacto (si diferente al anterior)

Ciudad

Provincia

Código postal IV. Datos del la Entidad que Gestiona las Actividades del Centro Coordinador (si diferente al anterior)

Denominación

Institut de Recerca de l’Hospital de la Santa Creu i Sant Pau

Domicilio

Sant Antoni Mª Claret, 167 Ciudad

Barcelona

Provincia Barcelona

Nombre y apellidos del representante legal Jordi Colomer Mascaró

(2)

V. Investigador Principal Coordinador Nombre

Juan

1º apellido Cinca

2º apellido Cuscullola DNI/Pasaporte

39.286.529-E

Titulación

Catedrático, Director del Servicio de Cardiología

Dirección de contacto Sant Antoni Mª Claret, 167 Ciudad

Barcelona

Provincia Barcelona

Código postal 08025

Teléfono 93.556.59.45 Fax

93.556.56.03

Correo electrónico jcinca@santpau.es

Número de miembros que componen el equipo del

Investigador Principal Coordinador 4

VI. Relación de Sub-proyectos que componen el Proyecto Principal (Añadir tantas tablas como sub-proyectos forman parte del proyecto principal)

Sub-proyecto I Molecular bases of adenosine receptor remodeling in human atrial fibrillation Datos del Centro Participante

Nombre

Universidad de Barcelona

Dirección Gran Vía, 585

Entidad privada Entidad pública Ciudad

Barcelona

Provincia Barcelona

País España Datos del Investigador Principal Participante

Nombre Rafael

1º apellido Franco

2º apellido Fernandez Dirección de contacto (si diferente al

anterior) Diagonal, 645

Ciudad Barcelona

Provincia Barcelona

Código postal 08028 País

España

Denominación del Cargo

Doctor en Química (Bioquímica)

Sub-proyecto II Functional cellular bases of adenosine receptor remodeling in human atrial fibrillation

Datos del Centro Participante Nombre

Dirección

Sant Antoni Mª Claret, 167

Barcelona

Provincia Barcelona

Nombre Leif

1º apellido Madsen

2º apellido Hove Dirección de contacto (si diferente al

anterior)

Lab.fisiologia Celular (St.antoni Mª Claret,167)

Ciudad Barcelona

Provincia Barcelona

Código postal 08025

País Denominación del Cargo

(3)

Sub-proyecto III Mathematical Modeling of Arrythmogenesis in the Human Atrium Datos del Centro Participante

Nombre

Universitat Politécnica de Catalunya

Dirección Jordi Girona, 31

Barcelona

Provincia Barcelona

Nombre Blas

1º apellido Echebarria

2º apellido Domínguez Dirección de contacto (si diferente al

anterior)

44-50 Avda. Dr. Marañón, EPSEB

Ciudad Barcelona

Provincia Barcelona

España

Denominación del Cargo Investigador Ramón y Cajal

Sub-proyecto IV In vivo validation of adenosine receptor remodeling in experimental atrial fibrillation

Datos del Centro Participante Nombre

Dirección

Barcelona

Provincia Barcelona

Nombre Juan

1º apellido Cinca

2º apellido Cuscullola Dirección de contacto (si diferente al

anterior)

Ciudad Barcelona

Provincia Barcelona

España

Denominación del Cargo

Catedrático, Director del Servicio de Cardiología

Fecha:

27 de junio de 2007

Juan Cinca

Firma del Investigador Principal Coordinador

27 de junio de 2007

Jordi Colomer

Firma y sello del Responsable Legal del Centro Coordinador.

27 de junio de 2007

Jordi Colomer

Firma y sello del Responsable Legal la Fundación

(4)

Financiación de Proyectos de Investigación Traslacional

Documento B: Memoria Proyecto

I. Datos Generales Número de Registro:

Título del Proyecto: Remodeling of adenosine receptors in human atrial fibrillation: A novel mechanism for atrial arrhythmogenesis

Denominación del Centro Coordinador: Hospital de la Santa Creu i Sant Pau Nombre y apellidos del Investigador Principal Coordinador: Juan Cinca Cuscullola Número de Grupos Participantes: 4

II. Resumen Científico del Proyecto

El resumen debe incluir : Objetivos, metodología e hipótesis de trabajo (500 palabras max.)

Hypothesis

Atrial fibrillation induces remodeling of cell membrane adenosine receptors and this in turn affects intracellular calcium handling and favours atrial arrhythmogenesis

Objectives

1. To investigate the molecular bases of atrial adenosine receptors remodeling in human atrial fibrillation (adenosine A1 and A2A expression, distribution and effect on phosphorylation of related calcium handling structures)

2. To investigate the electrophysiological bases atrial adenosine receptors remodeling in human atrial fibrillation (adenosine A1 and A2A modulation of calcium handling and effects on electrical and mechanical cell alternation)

3. To develop mathematical modeling of intracellular calcium handling and electrical alternation

4. To validate in an in vivo porcine model of atrial fibrillation the adenosine receptor remodeling concept

Methods

The molecular and electrophysiologic adenosine receptor studies will be conducted respectively in right atrial tissue and in isolated right atrial myocytes obtained during cardiac surgery in patients with and without atrial fibrillation. Western blot,

immunocytochemistry, second messengers determination, cell patch-clamp and confocal microscopy will be used.

The in vivo validation of the adenosine remodeling concept will be undertaken in

anesthetized pigs submitted to two different atrial fibrillation models (rapid atrial pacing and rapid ventricular pacing). Molecular and electrophysiological cell studies will be done to confirm the human data. To stablish the time relationship between the duration of the arrhythmia and the occurrence of adenosine receptor remodeling programmed electrical induction of atrial fibrillation will be used. Moreover, epicardial high density electrical mapping and rate-dependent electrical alternans will be performed to elucidate electrophysilogical arrhytmogenic mechanisms.

(5)

III. Memoria Científica del Proyecto

La memoria debe incluir los siguientes epígrafes:

- Introducción general al tema de investigación (1000 palabras max.) - Objetivos generales (500 palabras max.)

- Plan de trabajo (500 palabras max.) - Cronograma general (una página)

Atrial fibrillation (AF) is the most common cardiac arrhythmia. Epidemiological data depict that this arrhythmia affects about 5-6% of the general population older than 65 years and causes a fourfold increase of 4-year mortality in elderly people with heart failure. Moreover, systemic embolism is 5 times greater in these patients and it accounts for 15-20% of all systemic embolic episodes.

A characteristic feature of atrial fibrillation is the propensity of the arrhythmia to self- maintenance. Indeed, current clinical practice shows that the successful reversion rate of acute AF is inversely related to the previous duration of the arrhythmic episode and experimental models in awake goats settled the concept of domestication of atrial fibrillation as the arrhythmia episode lasted longer. The mechanism by which AF begets AF has been clearly linked to the development of a concurrent remodeling of the atrial electrophysiological properties taking place shortly after the onset of the arrhythmia episode. Chief among the electrophysiological disturbances found in remodeled fibrillating atria is the shortening of atrial refractoriness which favors re-entry by shortening the activation wave length. At the cellular level, shortening of the refractory period has been related to the lower density of potassium currents I_to and I_Kur and the L-type calcium current (I_Ca) as has been found in isolated atrial myocytes from patients with AF. Despite the important role of shortened atrial refractoriness in AF maintenance, other factors must play a role since shortening of atrial refractoriness is maximal within 2 days after the onset of AF, whereas permanent AF is only established after induced arrhythmia periods lasting longer than 2 weeks in animal models.

Factors that play a role in atrial remodelling are among others, related to abnormal intracellular calcium handling. Thus, isolated human atrial myocytes from patients with chronic AF show down-regulation of I_Ca amplitude and facilitation. Moreover, we have documented an abnormally high spontaneous calcium release from the sarcoplasmic reticulum also in human atrial myocytes that is likely caused by a concurrent hyper- phosphorylation of the sarcoplasmic reticulum calcium release channel (ryanodine receptor). Therefore, G_s-protein coupled membrane receptors that promote cAMP- dependent phosphorylation, such as the adenosine A_2A receptors (A_2AR), have the potential to promote abnormal calcium release from the sarcoplasmic reticulum and AF maintenance. Recent data from our laboratory confirm that A2ARs are expressed in human atrial myocytes and these receptors modulate spontaneous calcium release from the sarcoplasmic reticulum.

Therefore, this project aims to test the hypothesis that atrial electrical remodeling in patients with AF expands beyond the ionic channels and involves G-protein coupled receptors that contribute to AF maintenance through regulation of the phosphorylation- state of key calcium handling proteins in human atrial myocytes. Understanding of the pathophysiological role of adenosine receptor remodeling in human atrial fibrillation would settle the bases for research on novel therapeutical approaches of this arrhythmia.

(6)

HYPOTHESIS

Atrial fibrillation induces remodeling of cell membrane adenosine A1 and A2A receptors which in turn would promote abnormal phosphorylation of key calcium handling proteins and favor atrial arrhythmogeneicity

OBJECTIVES Main purpose

To test the working hypothesis this proposal will conduct molecular, electrophysiological, and mathematical modeling studies on human atrial fibrillation and then validate the concept of adenosine receptor remodeling in an in vivo porcine model of atrial fibrillation. For ethical reasons a human model of experimentally induced atrial fibrillation is not conceivable.

Specific objectives

1 To investigate the molecular bases of atrial adenosine receptors remodeling in human atrial fibrillation

2 To investigate the electrophysiological bases atrial adenosine receptors remodeling in human atrial fibrillation

3 To develop mathematical modeling of intracellular calcium handling and electrical alternation

4 To validate in an in vivo porcine model of atrial fibrillation the adenosine receptor remodeling concept

WORK PLAN

To achieve the specific objectives, the study is divided into 4 subprojects which will be conducted by expert research groups

Subproject # 1

Leader: Department of Biochemistry and Molecular Biology, University of Barcelona Activity: Investigate the molecular bases of adenosine receptor remodeling in human atrial fibrillation

Tasks:

1.1 To study the expression and distribution of adenosine A1, and A2A, and the calcium handling proteins SERCA2a, RyR-2 and CaV1.2 as well as the regulatory protein PLB in human and porcine atrial samples from normal and fibrillating atria.

1.2 To study the effect of adenosine receptor activation on phosphorylation of L-type calcium channels, phospholamban and the ryanodine receptors in normal and fibrillating human and porcine atria. We shall examine and compare PKA and CaMKII mediated phosphorylation

Subproject #2

Leader: Cell Physiology Laboratory, Cardiology Department, Hospital de la Santa Creu i Sant Pau, Autonomous University of Barcelona

Activity: To investigate he functional cellular bases of adenosine receptor remodeling in human atria fibrillation

Tasks:

2.1 Investigate adenosine receptor mediated modulation of calcium handling at rest and the beat-to-beat response in human and porcine atrial myocytes from normal and fibrillating atria. The specific roles of PKA and CaMKII mediated phosphorylation will be addressed.

(7)

2.2 Determine the effect of adenosine A1, and A2A receptor activation and inhibition of the relationship between the rate-dependent beat-to-beat responses of intracellular calcium, membrane potential and cell shortening in atrial myocytes.

2.3 Investigate how cellular adenosine metabolism modulates calcium handling and the rate-dependent beat-to-beat response in human atrial myocytes from patients with and without AF.

Subproject #3

Leader: Department of Applied Physics, Universitat Politecnica de Catalunya, Barcelona

Activity: To develope a mathematical modeling of arrhythmogenesis in the human atrium

Tasks:

3.1 To develop a single human atrial myocyte model that adequately describes intracellular calcium handling and cell shortening. The model will incorporate measurements from isolated human atrial myocytes.

3.2 To use a 2-D model of atrial sheet, incorporating differences in the rate-dependent beat-to-beat response among individual myocytes, to predict the effect of global and local modulation of specific calcium handling mechanisms on the rate-dependent response of intracellular calcium handling.

Subproject # 4

Leader: The Integrative Cardiac Physiology Laboratory, Cardiology Department, Hospital de la Santa Creu i Sant Pau, Autonomous University of Barcelona

Activity: In vivo validation of adenosine receptor remodeling in experimental atrial fibrillation

Tasks:

4.1 To evaluate the role of adenosine A2A receptors in the pathophysiology of electrical remodeling in a porcine model of AF induced by rapid atrial pacing.

4.2 To evaluate the presence of adenosine A2A receptors in the CHF model of AF where structural remodeling is the main component and electrical remodeling is almost absent.

Fecha y firma del Investigador Principal Coordinador 27 junio 2007

Juan Cinca