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Induction of apoptosis in cardiac myocytes by an A3 adenosine receptor agonist
V Shneyvays1, H Nawrath, K A Jacobson
1Department of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel.
Abstract:
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.
Insights
Activation of the A3 adenosine receptor by IB-MECA induces apoptosis in rat cardiomyocytes, causing nuclear changes and contractile dysfunction. These effects are reversible, suggesting a role for A3 receptor activation in cardiac cell death.
Area of Science:
- Cardiovascular Biology
- Molecular Pharmacology
- Cell Death Mechanisms
Background:
- Adenosine (ADO) receptors, particularly A1 and A3 subtypes, play roles in cardiac function.
- The specific role of A3 adenosine receptor activation in cardiomyocyte apoptosis remains to be fully elucidated.
Purpose of the Study:
- To investigate the effects of the selective adenosine A3 receptor agonist IB-MECA on cultured newborn rat cardiomyocytes.
- To compare these effects with adenosine and selective agonists/antagonists of other adenosine receptor subtypes.
- To determine the impact of A3 receptor activation on cardiomyocyte apoptosis, contractility, and intracellular calcium levels.
Main Methods:
- Cultured newborn rat cardiomyocytes were treated with IB-MECA, adenosine, R-PIA (A1 agonist), or MRS 1191 (A3 antagonist).
- Digital image analysis of Feulgen-stained nuclei was used to assess apoptosis.
- In situ nick end labeling (TUNEL assay) identified DNA breaks.
- Light microscopy monitored cell contraction and beat frequency.
- Intracellular free calcium concentration ([Ca2+]i) was measured.
Main Results:
- High concentrations of IB-MECA and adenosine induced apoptosis in cardiomyocytes, characterized by nuclear chromatin condensation and breakdown.
- IB-MECA treatment led to disordered contraction waves, decreased beat frequency, and reduced numbers of contracting cells in a dose-dependent manner.
- IB-MECA caused a rapid, sustained increase in intracellular calcium ([Ca2+]i), followed by cessation of beating, with full recovery observed post-treatment.
Conclusions:
- Activation of the A3 adenosine receptor by IB-MECA can induce apoptosis in cardiomyocytes.
- A3 receptor activation disrupts cardiomyocyte contractility and intracellular calcium homeostasis.
- These findings suggest a potential role for A3 adenosine receptor signaling in the process of cardiac cell apoptosis.