Productos de Laboratorio

Research Project

Summary

Introduction: The extent of myocardial necrosis occurring during acute coronary syndrome is the main determinant of survival and quality of life in patients with ischemic heart disease. Besides the ischemic effects per se, conditions in which recanalization or reperfusion occurs are also the keys to determine the final extent of injury. In spite of the increasing knowledge about the mechanisms of cell death during reperfusion and its clinical importance, until now treatments assayed with success at the experimental level have not could to be used in patients: because of their toxic effects, limited therapeutic range or the ignorance of the long-term effects. In the last years, it has been demonstrated that the normalization of intramyocardial cyclic GMP levels (cGMP), depleted during ischemia-reperfusion, limits reperfusion-injury.

Figure 1. Endothelial cells from rat heart in culture.

Objective: The interest of the present project is to analyze the possible therapeutic usefulness of the modulation of NO/cGMP-transduction pathway in ischemic heart disease.

Methodology: Three different models will be used: myocardial cells in culture, isolated rat heart and in situ pig heart.

Design: Mechanisms causing cGMP depletion during ischemia-reperfusion and cGMP effects (protection/apoptosis?) on reperfusion-injury will be analyzed in the two first phases of the study. In the second phase, new pharmacological dianas in cGMP pathway will be also investigated. Those ones with therapeutic potential will be tested in the last phase of the study in in situ pig heart.

Figure 2. Isolated cardiomyocytes (cardiac muscle cells) from rat heart.

Objectives

Ischemic heart disease is the leading cause of death in Europe. The extent of myocardial necrosis occurring during acute coronary syndrome is the main determinant of survival and quality of life in patients with ischemic heart disease. On the other hand, there is increasing evidence that necrosis is only partly due to ischemic conditions themselves and that reperfusion aggravates the extent of injury. The possibility of modulating the reperfusion conditions to decrease myocardial necrosis is of special therapeutic interest because it frequently takes place in the clinical environment.

The main mechanism of cellular death during the first minutes of reperfusion is hypercontracture of myofibrils (consequence of the restoration of ATP levels in the presence of elevated intracellular Ca²⁺ concentrations). Systems regulating the increase in [Ca²⁺]i during ischemia-reperfusion play a crucial role in the extent of necrosis: Na⁺/H⁺-exchanger (increasing intracellular Na⁺ levels), Na⁺/Ca²⁺-exchanger (allowing Ca²⁺ entrance) and gap-junctions (which allow cell-to-cell propagation of the Na⁺ increase) [1,2]. Although several substances have been able to limit reperfusion-injury at the experimental level, application to patients have not been possible until now, among other reasons, because of their narrow therapeutic range, toxicity, and the absence of knowledge about their long-term effects.

Figure 3. (a) Isolated rat heart, (b) In situ pig heart.

In the last years, the protective role on reperfusion-injury of increasing cGMP synthesis (impaired during ischemia-reperfusion) has been demonstrated in different experimental models [3,4]. In particular, the administration of the natriuretic peptide urodilatin has been found to be protective at very low doses when it is added at the same time of reperfusion [5,6]. This substance has also interest because it is used in humans to treat heart failure without important secondary effects.

Causes of the decrease in the myocardial content of cGMP during ischemia-reperfusion and the precise mechanism of the protective effect of cGMP during reperfusion are not known. cGMP is able to decrease early reperfusion-injury at least partly due to the reduction of myocardial hypercontracture. cGMP has been described to modulate Na⁺/H⁺- and Na⁺/Ca²⁺-exchangers and to reduce gap-junction permeability. cGMP also modulates Ca²⁺-channels, vascular permeability and cell adhesion to the endothelium. It is yet unknown, which of these potential cGMP effects acts during reperfusion.

On the other hand, there are two findings that may limit the therapeutic application of urodilatin: urodilatin present a biphasic protective effect [5,6] and it has been described that cGMP can stimulate apoptotic death [7].

According to the previous data, we propose the following objectives:

First objective: To study the mechanisms involved in the decrease of the cGMP synthetic capability evoked by ischemia-reperfusion.

Second objective: To analyze the possible mechanisms mediating the cGMP protective effect on hypercontracture (PKG, gap-junctions,...) and to look for alternative pharmacological dianas to particulate guanylyl cyclase (mGC) stimulated by urodilatin.

Third objective: To study the role of cGMP in the apoptosis induction during reperfusion and the implicated mechanisms.

Fourth objective: To study the applicability to patients with myocardial infarction of those modulators of cGMP transduction pathway presenting therapeutic potential in the previous phases of the project.

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[2] Ruiz-Meana M, Garcia-Dorado D, Hofstaetter B et al. Circ.Res. 1999,85:280
[3] Agulló L, Garcia-Dorado D, Inserte J et al. Am.J.Physiol. 1999,276:H1574
[4] Padilla F, Garcia-Dorado D, Agulló L et al. Cardiovasc.Res. 2000,46:412
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