Role of adenosine A1 and A3 receptors in regulation of cardiomyocyte homeostasis after mitochondrial respiratory chain injury

Abstract

Activation of either the A(1) or the A(3) adenosine receptor (A(1)R or A(3)R, respectively) elicits delayed cardioprotection against infarction, ischemia, and hypoxia. Mitochondrial contribution to the progression of cardiomyocyte injury is well known; however, the protective effects of adenosine receptor activation in cardiac cells with a respiratory chain deficiency are poorly elucidated. The aim of our study was to further define the role of A(1)R and A(3)R activation on functional tolerance after inhibition of the terminal link of the mitochondrial respiratory chain with sodium azide, in a state of normoxia or hypoxia, compared with the effects of the mitochondrial ATP-sensitive K(+) channel opener diazoxide. Treatment with 10 mM sodium azide for 2 h in normoxia caused a considerable decrease in the total ATP level; however, activation of adenosine receptors significantly attenuated this decrease. Diazoxide (100 muM) was less effective in protection. During treatment of cultured cardiomyocytes with hypoxia in the presence of 1 mM sodium azide, the A(1)R agonist 2-chloro-N(6)-cyclopentyladenosine was ineffective, whereas the A(3)R agonist 2-chloro-N(6)-iodobenzyl-5'-N-methylcarboxamidoadenosine (Cl-IB-MECA) attenuated the decrease in ATP level and prevented cell injury. Cl-IB-MECA delayed the dissipation in the mitochondrial membrane potential during hypoxia in cells impaired in the mitochondrial respiratory chain. In cells with elevated intracellular Ca(2+) concentration after hypoxia and treatment with NaN(3) or after application of high doses of NaN(3), Cl-IB-MECA immediately decreased the elevated intracellular Ca(2+) concentration toward the diastolic control level. The A(1)R agonist was ineffective. This may be especially important for the development of effective pharmacological agents, because mitochondrial dysfunction is a leading factor in the pathophysiological cascade of heart disease.

Fig. 1

Effects of adenosine receptor (AR) activation on lactate dehydrogenase (LDH) release from cardiomyocytes treated with 1 mM sodium azide and exposed to hypoxia for 90 or 60 min. The A₁ AR subtype (A₁R) agonist 2-chloro-N⁶-cyclopentyladenosine (CCPA, 100 nM) or the A₃ AR subtype (A₃R) agonist 2-chloro-N⁶-iodobenzyl-5′-N-methylcarboxamidoadenosine (Cl-IB-MECA, 100 nM) were given 15 min before the insults. LDH release was determined immediately after hypoxia. Release in the control cultures was considered to be 100%. *P < 0.05; **P < 0.01 compared with hypoxia, according to ANOVA and a post hoc Tukey-Kramer test.

Fig. 2

Effects of activation of ARs on cardiomyocyte death. Effects of the A₁R agonist CCPA (100 nM) and A₃R agonist Cl-IB-MECA (100 nM) were studied after treatment of the cells with sodium azide (1 mM) and exposure to hypoxia for 60 or 90 min. *P < 0.01 compared with cells treated with sodium azide and hypoxia.

Fig. 3

Effects of AR activation on ATP levels in cardiomyocytes treated with 1 mM sodium azide and exposed to hypoxia for 90 or 60 min. Effects of the A₁R agonist CCPA (100 nM) or the A₃R agonist Cl-IB-MECA (100 nM) were studied on ATP levels in cell homogenates. *P < 0.05; **P < 0.01 compared with hypoxia.

Fig. 4

Effects of AR activation and ATP-sensitive K⁺ (K_ATP) channels on ATP levels in cardiomyocytes treated for 2 h with 10 mM sodium azide. Effects of the A₁R agonist CCPA (100 nM), the A₃R agonist Cl-IB-MECA (100 nM), the A₁R antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX, 1 µM), the A₃R antagonist 5-propyl-2-ethyl-4-propyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate (MRS-1523, 1 µM), and the mitochondrial K_ATP channel blocker 5-hyrdoxydecanoate (5-HD, 0.3 mM) were studied on ATP levels in cultured cardiomyocytes. *P < 0.05; **P < 0.01 compared with cells treated with sodium azide.

Fig. 5

Effects of A₁R and A₃R activation on 4-[4-(dimethylamino)styryl]-N-methylpyridinium iodide (DASPMI) fluorescence in mitochondria of cardiomyocytes treated with sodium azide. Four-day-old cardiomyocytes were exposed to 10 mM sodium azide for 120 min. A: control cells. B: treatment with 10 mM NaN₃ in the absence of Cl-IB-MECA. C: treatment with 10 mM NaN₃ in the presence of 100 nM Cl-IB-MECA. D: treatment with 10 mM NaN₃ and 100 nM CCPA. E and F: DASPMI fluorescence in mitochondria of cardiomyocytes treated with sodium azide (1 mM) and exposed to hypoxia for 60 min (E) or 90 min (F). G and H: DASPMI fluorescence during 60 min of hypoxia and sodium azide (1 mM) shows the effects of A₃R activation with 100 nM Cl-IB-MECA (G) and the effects of A₁R activation with 100 nM CCPA (H). Bar = 10 µm. Each image is representative of six experiments.

Fig. 6

Changes in mitochondrial membrane potential (Δψ) after AR activation. A: sodium succinate (10 mM) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, 5 µM) were applied as standards for mitochondrial energy generation and dissipation. B: effects of A₁R agonist CCPA (100 nM), A₃R agonist Cl-IB-MECA (100 nM), and mitochondrial K_ATP channel opener diazoxide (100 µM) on DASPMI fluorescence during normoxia. C and D: A₁R agonist CCPA (100 nM) and A₃R agonist Cl-IB-MECA (100 nM), respectively, were effective in retarding a decrease in DASPMI fluorescence and, hence, dissipation of Δψ during hypoxia. Pretreatment of the cells with DPCPX (1 µM) before addition of CCPA or with MRS-1523 (1 µM) before addition of Cl-IB-MECA abolished the protective effects of these agonists. E: effects of AR activation and diazoxide on kinetics of Δψ. Effects of the A₁R agonist CCPA (100 nM), A₃R agonist Cl-IB-MECA (100 nM), and diazoxide (100 µM) on DASPMI fluorescence in cardiomyocytes treated with sodium azide (1 mM) and exposed to hypoxia. Readings were obtained every 10 min. Each graph is representative of six experiments.

Fig. 7

Effects of AR activation on intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cultured cardiomyocytes. A: sodium azide (10 mM) induced transient accelerations of the beating rate, elevation of diastolic [Ca²⁺]_i, and termination of beating activity after 1.5–2 h of treatment. B: pretreatment of cultures with 100 nM Cl-IB-MECA abolished [Ca²⁺]_i elevation after treatment with azide and maintained myocyte contractility. C: averaged data obtained from six experiments. Treatment of cultured cardiac muscle cells with 10 mM NaN₃ led to elevation of baseline [Ca²⁺]_i and disappearance of [Ca²⁺]_i transient amplitude. Cl-IB-MECA (100 nM) restricted elevation of baseline [Ca²⁺]_i (*P < 0.05 vs. NaN₃ group; n = 18 cells) and maintained muscle cell contractility (**P < 0.01 vs. NaN₃ group; n = 18 cells). D: continuous monitoring of [Ca²⁺]_i during hypoxia (Ar) in cultures pretreated with 1 mM NaN₃. E: application of 100 nM Cl-IB-MECA after increase of the basal level [Ca²⁺]_i returned it to normal diastolic level, and beating activity was restored. F: A₁R agonist CCPA (100 nM) in the same experiment was ineffective. G: application of Cl-IB-MECA (100 nM) for 15 min before sodium azide application (NaN₃) maintained contractile activity and Ca²⁺ oscillations during 40–60 min. H: protective effect of A₁R agonist CCPA (100 nM) was observed during 15–20 min of hypoxia after application of 1 mM NaN₃. I: averaged data obtained from six experiments. Exposure of cultured cardiomyocytes to hypoxia with 1 mM NaN₃ led at 40 min to elevation of baseline [Ca²⁺]_i and disappearance of [Ca²⁺]_i transient amplitude. Pretreatment with Cl-IB-MECA restricted elevation of baseline [Ca²⁺]_i (*P < 0.05 vs. NaN₃ group; n = 18 cells) and maintained muscle cell contractility (**P < 0.01 vs. NaN₃ group; n = 18 cells).