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

Abstract

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.

Figure 1

Effects of activation of A₁R and A₃R on DOX-induced LDH release. Four days old cardiomyocyte cultures were exposed to DOX for 18 h, then incubated for 24 h in drug-free medium. A₁R agonist CCPA (100 nm) or A₃R agonist Cl-IB-MECA (100 nm) were introduced to the medium 10 min before DOX administration and left in culture during DOX treatment. A₃R selective antagonist MRS1523 (1 μm) was added to medium 10 min before Cl-IB-MECA application. The significance of observed differences was determined by ANOVA. Means with the same letter are not significantly different (n=6, P<0.05) according to a post-hoc Tukeys test.

Figure 2

Effect of adenosine A₁R and A₃R activation on intracellular Ca²⁺ concentration in cultured cardiomyocytes treated with DOX. (A) Stability of intracellular calcium level and [Ca²⁺]_i transients in control cardiomyocytes. (B) Effects of A₃R agonist Cl-IB-MECA (0.1–1 μm) on intracellular calcium levels in cultured cardiomyocytes. (C) Effects of A₁R agonist CCPA (0.1–1 μm) on intracellular calcium level. (D) Progressive slow elevation of the intracellular calcium and abnormality in oscillations after DOX (5 μm) treatment. (E) Attenuation of Ca²⁺ elevation after DOX (5 μm) treatment by Cl-IB-MECA (100 nm). (F) Prevention of protective effect of Cl-IB-MECA by A₃R antagonist MRS1523 (1 μm). Ligands were added to the medium before treatment with DOX and left during the treatment. (G) The A₁R agonist CCPA (100 nm) was not effective in prevention of intracellular calcium elevation in cardiac cells after DOX (5 μm) treatment. Drugs were added at the indicated time (arrows).

Figure 2

Effect of adenosine A₁R and A₃R activation on intracellular Ca²⁺ concentration in cultured cardiomyocytes treated with DOX. (A) Stability of intracellular calcium level and [Ca²⁺]_i transients in control cardiomyocytes. (B) Effects of A₃R agonist Cl-IB-MECA (0.1–1 μm) on intracellular calcium levels in cultured cardiomyocytes. (C) Effects of A₁R agonist CCPA (0.1–1 μm) on intracellular calcium level. (D) Progressive slow elevation of the intracellular calcium and abnormality in oscillations after DOX (5 μm) treatment. (E) Attenuation of Ca²⁺ elevation after DOX (5 μm) treatment by Cl-IB-MECA (100 nm). (F) Prevention of protective effect of Cl-IB-MECA by A₃R antagonist MRS1523 (1 μm). Ligands were added to the medium before treatment with DOX and left during the treatment. (G) The A₁R agonist CCPA (100 nm) was not effective in prevention of intracellular calcium elevation in cardiac cells after DOX (5 μm) treatment. Drugs were added at the indicated time (arrows).

Figure 3

Effects of activation of A₁R and A₃R on DOX-induced intensification of lipid peroxidation. Four days old cardiomyocytes in vitro were exposed to DOX for 18 h, then left for 24 h in drug-free medium. The level of lipid hydroperoxide (LOOH) was estimated after exposure to 0.5–5 μm DOX. The A₁R agonist CCPA (100 nm), A₃R agonist Cl-IB-MECA (100 nm) and A₃R antagonist MRS1523 (1 μm) were added to the medium before treatment with DOX and left during the treatment. Significance of the differences observed was determined by ANOVA. Means with the same letter are not significantly different (n=6, P<0.05) according to a post-hoc Tukey’s test.

Figure 4

Light microscopic observation of cytochrome C oxidase (COX) activity in cultured cardiomyocytes. Effects of activation of A₃R on DOX-induced alterations in COX activity. Four days old cardiomyocytes in vitro were exposed to DOX for 18 h, then incubated for 24 h in drug-free medium. A₃R agonist Cl-IB-MECA (100 nm) was added to medium before treatment with DOX. (A) Control cells. Dark COX-positive mitochondria revealed in the perinuclear and intermyofibrillar regions. (B) Cells treated with Cl-IB-MECA (100 nm). (C) Decrease in COX activity after 0.5 μm DOX treatment. (D) Considerable decrease in COX activity in cells treated with 2 µm DOX. (E) Protection of COX activity in cells treated with 0.5 μm DOX by activation of A₃R with Cl-IB-MECA. (F) Moderate protection of COX activity in cells treated with 2 μm DOX by activation of A₃R with Cl-IB-MECA (bars=10 μm). (G) Definition of the ratio of COX-positive area in the image of stained cells to the total image area. The significance of observed differences was determined by ANOVA. Means with the same letter are not significantly different (n=6, P<0.05) according to a post-hoc Tukey’s test.

Figure 5

Effects of the activation of A₁R and A₃R on the ATP level in cultured cardiomyocytes after DOX treatment. Four days old cardiomyocytes in vitro were exposed to DOX for 18 h, then left for 24 h in drug-free medium. A₁R agonist CCPA (100 nm) and A₃R agonist Cl-IB-MECA (100 nm) were added to the medium 10 min before DOX administration and left in the culture during DOX treatment. A₃R selective antagonist MRS1523 was added to the media 10 min before Cl-IB-MECA application. The significance of observed differences was determined by ANOVA. Means with the same letter are not significantly different (n=6, P<0.05) according to a post-hoc Tukey’s test.