Acute, nongenomic effect of thyroid hormones in preventing calcium overload in newborn rat cardiocytes.

In this study, we examined the acute effects of thyroid hormones (TH) T(3) and T(4), leading to improvement of myocardial function through activation of Ca(2+) extrusion mechanisms and, consequently, prevention of intracellular calcium overload. Extracellular calcium elevation from 1.8 to 3.8 mM caused immediate increase in intracellular calcium level ([Ca(2+)](i)) in newborn cardiomyocyte cultures. Administration of 10 or 100 nM T(3) or T(4) rapidly (within 10 sec) decreased [Ca(2+)](i) to its control level. Similar results were obtained when [Ca(2+)](i) was elevated by decreasing extracellular Na(+) concentration, causing backward influx of Ca(2+) through Na(+)/Ca(2+) exchanger, or by administration of caffeine, releasing Ca(2+) from the sarcoplasmic reticulum (SR). Under these conditions, T(3) or T(4) decreased [Ca(2+)](i). T(3) and T(4) also exhibited protective effects during ischemia. T(3) or T(4) presence during hypoxia for 120 min in culture medium restricted the increase of [Ca(2+)](i) and prevented the pathological effects of its overload. An inhibitor of SR Ca(2+)-ATPase (SERCA2a), thapsigargin, increases [Ca(2+)](i) and in its presence neither T(3) nor T(4) had any effect on the [Ca(2+)](i) level. The reduction of [Ca(2+)](i) level by T(3) and T(4) was also blocked in the presence of H-89 (a PKA inhibitor), and by calmodulin inhibitors. The effect of TH on the reduction of [Ca(2+)](i) was prevented by propranolol, indicating that the hormones exert their effect through interaction with adrenergic receptors. These results support our hypothesis that TH prevent calcium overload in newborn rat cardiomyocytes, most likely by a direct, acute, and nongenomic effect on Ca(2+) transport into the SR.

Angiotensin II-induced apoptosis in rat cardiomyocyte culture: a possible role of AT1 and AT2 receptors.

OBJECTIVES: To investigate the mechanism of angiotensin II-induced apoptosis in cultured cardiomyocytes by determining which receptor subtype is involved, and what is the relationship between intracellular Ca2+ changes and apoptosis. DESIGN AND METHODS: Neonatal rat cardiomyocytes were pretreated with either the AT1 antagonist irbesartan or the AT2 antagonist PD123319 before exposure to angiotensin II. Apoptosis was evaluated using morphological technique, staining nuclei by Feulgen and Hoechst methods followed by image analysis and by in situ terminal deoxynucleotidyl transferase nick-end (TUNEL) labelling. TUNEL-positive cardiocytes were distinguished from other cells by double staining with alpha-sarcomeric actin. Intracellular Ca2+ changes were assessed by indo-1 fluorescence microscopy, and the effect of Ca2+ on angiotensin II-induced apoptosis was tested using the calcium channel blocker verapamil. RESULTS: Exposure to angiotensin II (10 nmol/l) resulted in cell replication and a three-fold increase in programmed cell death (P < 0.05). Pretreatment with either irbesartan (an AT1receptor antagonist, 100 nmol/l) or PD123319 (an AT2 receptor antagonist, 1 micromol/l) prevented the angiotensin II-induced apoptosis, indicating the presence of both AT1 and AT2receptors on cardiomyocytes. Exposure of myocytes to angiotensin II caused an immediate and dose-dependent increase in the concentration of intracellular free Ca2+ that lasted 40-60 s. The effect was sustained in a Ca2+ free medium. Pretreatment of cells with irbesartan (100 nmol/l) and PD123319 (10 micromol/l) blocked Ca2+ elevation. Pretreatment with verapamil (10 micromol/l) prevented angiotensin II-induced apoptosis. CONCLUSIONS: Angiotensin II-induced apoptosis in rat cardiomyocytes is mediated through activation of both AT1 and AT2 receptors. The apoptotic mechanism is not related to the immediate angiotensin II-induced Ca2+ rise from intracellular stores. However, it is accompanied by cardiomyocyte proliferation and requires Ca2+ influx through L-type channel activity.

Activation of A(3)adenosine receptor protects against doxorubicin-induced cardiotoxicity.

Adenosine exerts a marked protective effect on the heart during cardiac ischemia. This protection is mediated by binding to the A(1)and A(3)subtypes of adenosine receptor (A(1)R and A(3)R, respectively). The objective of the present study was to investigate whether activation of A(1)and A(3)adenosine receptors may reduce doxorubicin-induced damage to cardiomyocytes in culture. Cultured cardiomyocytes from newborn rats were treated with 0.5--5 microm doxorubicin (DOX) for 18 h and then incubated in drug-free medium for an additional 24 h. This treatment resulted in cell damage and lactate dehydrogenase release, even after low (0.5 microm) doses of the drug, and increased in a concentration-dependent manner. Activation of A(3)-subtype but not A(1)-subtype receptors attenuated doxorubicin-cardiotoxicity after drug treatment for 18 h followed by 24 h incubation in drug-free medium. Modulation of intracellular calcium mediated by activation of A(3)R, but not by A(1)R, in cultured myocytes suggested an important pathophysiological significance of this subtype of adenosine receptors. Protection by A(3)R agonist Cl-IB-MECA (2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide) following DOX treatment is evident in: (1) decreases in intracellular calcium overloading and abnormalities in Ca(2+)transients; (2) reduction of free-radical generation and lipid peroxidation; (3) attenuation of mitochondrial damage by protection of the terminal link (COX-complex) of respiratory chain; (4) attenuation of the decrease in ATP production and irreversible cardiomyocyte damage. Cardioprotection caused by Cl-IB-MECA was antagonized considerably by the selective A(3)adenosine receptor antagonist MRS1523.

Cardioprotective effects of adenosine A1 and A3 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 A1 and A3 receptors (A1R and A3R), 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 N2 (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 A1 and A3 receptor antagonists abolished the protective effects of adenosine. A1R and A3R 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 A1R agonist, CCPA, was abolished by an A1R antagonist, DPCPX, and was not affected by an A3R antagonist, MRS 1523. Cardioprotection caused by the A3R agonist, Cl-IB-MECA, was antagonized completely by MRS 1523 and only partially by DPCPX. Activation of both A1R and A3R together was more efficient in protection against hypoxia than by each one alone. Our study indicates that activation of either A1 or A3 adenosine receptors in the rat can attenuate myocyte injury during hypoxia. Highly selective A1R and A3R agonists may have potential as cardioprotective agents against ischemia or heart surgery.

Induction of apoptosis in rat cardiocytes by A3 adenosine receptor activation and its suppression by isoproterenol.

The purpose of the present study was to investigate the mechanisms involved in the induction of apoptosis in newborn cultured cardiomyocytes by activation of adenosine (ADO) A3 receptors and to examine the protective effects of beta-adrenoceptors. The selective agonist for A3 ADO receptors Cl-IB-MECA (2-chloro-N6-iodobenzyl-5-N-methylcarboxamidoadenosine) and the antagonist MRS1523 (5-propyl-2-ethyl-4-propyl-3-(ethylsulfanylcarbonyl)-6-phenylpy rid ine-5-carboxylate) were used. High concentrations of the Cl-IB-MECA (> or = 10 microM) agonist induced morphological modifications of myogenic cells, such as rounding and retraction of cell body and dissolution of contractile filaments, followed by apoptotic death. In addition, Cl-IB-MECA caused a sustained and reversible increase in [Ca2+]i, which was prevented by the selective antagonist MRS1523. Furthermore, MRS1523 protected the cardiocytes if briefly exposed to Cl-IB-MECA and partially protected from prolonged (48 h) agonist exposure. Apoptosis induced by Cl-IB-MECA was not redox-dependent, since the mitochondrial membrane potential remained constant until the terminal stage of cell death. Cl-IB-MECA activated caspase-3 protease in a concentration-dependent manner after 7 h of treatment and more effectively after 18 h of exposure. Bcl-2 protein was readily detected in control cells, and its expression was significantly decreased after 24 and 48 h of treatment with Cl-IB-MECA. Beta-adrenergic stimulation antagonized the pro-apoptotic effects of Cl-IB-MECA, probably through a cAMP/protein kinase A-independent mechanism, since addition of dibutyryl-cAMP did not abolish the apoptosis induced by Cl-IB-MECA. Incubation of cultured myocytes with isoproterenol (5 microM) for 3 or 24 h almost completely abolished the increase in [Ca2+]i. Prolonged incubation of cardiomyocytes with isoproterenol and Cl-IB-MECA did not induce apoptosis. Our data suggest that the apoptosis-inducing signal from activation of adenosine A3 receptors (or counteracting beta-adrenergic signal) leads to the activation of the G-protein-coupled enzymes and downstream pathways to a self-amplifying cascade. Expression of different genes within this cascade is responsible for orchestrating either cardiomyocyte apoptosis or its protection.

Characterization of "mini-nucleotides" as P2X receptor agonists in rat cardiomyocyte cultures. An integrated synthetic, biochemical, and theoretical study.

The design and synthesis of "mini-nucleotides", based on a xanthine-alkyl phosphate scaffold, are described. The physiological effects of the new compounds were evaluated in rat cardiac cell culture regarding Ca(2+) elevation and contractility. The results indicate biochemical and physiological profiles similar to those of ATP, although at higher concentrations. The biological target molecules of these "mini-nucleotides" were identified by using selective P2-R and A(1)-R antagonists and P2-R subtype selective agonists. On the basis of these results and of experiments in Ca(2+) free medium, in which [Ca(2+)](i) elevation was not observed, we concluded that interaction of the analogues is likely with P2X receptor subtypes, which causes Ca(2+) influx. Theoretical calculations analyzing electronic effects within the series of xanthine-alkyl phosphates were performed on reduced models at quantum mechanical levels. Calculated dipole moment vectors, electrostatic potential maps, and volume parameters suggest an explanation for the activity or inactivity of the synthesized derivatives and predict a putative binding site environment for the active agonists. Xanthine-alkyl phosphate analogues proved to be selective agents for activation of P2X-R subtypes, whereas ATP activated all P2-R subtypes in cardiac cells. Therefore, these analogues may serve as prototypes of selective drugs aiming at cardiac disorders mediated through P2X receptors.

Induction of apoptosis in cardiac myocytes by an A3 adenosine receptor agonist.

The effects of the selective adenosine (ADO) A3 receptor agonist IB-MECA (N6-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide) on cultured newborn rat cardiomyocytes were examined in comparison with ADO, the ADO A1 receptor-selective agonist R-PIA (N6-R-phenylisopropyladenosine), or the ADO A3 selective antagonist MRS 1191 (3-ethyl-5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1, 4-(+/-)-dihydropyridine-3,5 dicarboxylate), using digital image analysis of Feulgen-stained nuclei. At high concentration, IB-MECA (>/=10 microM ) and ADO (200 microM) induced apoptosis; however, R-PIA or MRS 1191 did not have any detectable effects on cardiac cells. In addition, DNA breaks in cardiomyocytes undergoing apoptosis following treatment by IB-MECA were identified in situ using the nick end labeling of DNA ("TUNEL"-like) assay. In the presence of >/=10 microM IB-MECA, disorder in the contraction waves appeared, and a decrease in the frequency of beats was observed. Analysis with light microscopy revealed that the number of contracting cells decreased in a concentration-dependent manner. The A3 receptor agonist IB-MECA caused an increase in intracellular free calcium concentration ([Ca2+]i). The drug produced a rapid rise followed by a sustained increase in [Ca2+]i, which lasted for 40-60 s. Finally, cessation of beating and Ca2+ transients were observed. Full recovery of contractile activity and rhythmical Ca2+ transients were observed 15-20 min after IB-MECA treatment. The induction of apoptosis in the cardiocytes by IB-MECA led to the appearance of features of apoptotic nuclei: the onset of condensation, compacting, and margination of nuclear chromatin. These effects were accompanied by the disintegration of the structural framework of the nucleus and nuclear breakdown. The results suggest that activation of the A3 adenosine receptor may participate in the process of apoptosis in cardiomyocytes.