Cardioprotective effects of adenosine A(1) and A(3) receptor activation during hypoxia in isolated rat cardiac myocytes

Adenosine (ADO) is a well-known regulator of a variety of physiological functions in the heart. In stress conditions, like hypoxia or ischemia, the concentration of adenosine in the extracellular fluid rises dramatically, mainly through the breakdown of ATP. The degradation of adenosine in the ischemic myocytes induced damage in these cells, but it may simultaneously exert protective effects in the heart by activation of the adenosine receptors. The contribution of ADO to stimulation of protective effects was reported in human and animal hearts, but not in rat hearts. The aim of this study was to evaluate the role of adenosine A(1) and A(3) receptors (A(1)R and A(3)R), in protection of isolated cardiac myocytes of newborn rats from ischemic injury. The hypoxic conditions were simulated by exposure of cultured rat cardiomyocytes (4-5 days in vitro), to an atmosphere of a N-2 (95%) and CO2 (5%) mixture, in glucose-free medium for 90 min. The cardiotoxic and cardioprotective effects of ADO ligands were measured by the release of lactate dehydrogenase (LDH) into the medium. Morphological investigation includes immunohistochemistry, image analysis of living and fixed cells and electron microscopy were executed. Pretreatment with the adenosine deaminase considerably increased the hypoxic damage in the cardiomyocytes indicating the importance of extracellular adenosine. Blocking adenosine receptors with selective A(1) and A(3) receptor antagonists abolished the protective effects of adenosine. A(1)R and A(3)R activation during the hypoxic insult delays onset of irreversible cell injury and collapse of mitochondrial membrane potential as assessed using DASPMI fluorochrom. Cardioprotection induced by the A(1)R agonist, CCPA, was abolished by an A(1)R antagonist, DPCPX, and was not affected by an A(3)R antagonist, MRS1523. Cardioprotection caused by the A(3)R agonist, Cl-IB-MECA, was antagonized completely by MRS1523 and only partially by DPCPX. Activation of both A(1)R and A(3)R together was more efficient in protection against hypoxia than by each one alone. Our study indicates that activation of either A(1) or A(3) adenosine receptors in the rat can attenuate myocyte injury during hypoxia. Highly selective A(1)R and A(3)R agonists may have potential as cardioprotective agents against ischemia or heart surgery.