Effects of cardiac-restricted overexpression of the A(2A) adenosine receptor on adriamycin-induced cardiotoxicity

Abstract

Activation of the A(2A) adenosine receptor (A(2A)R) has been shown to be cardioprotective. We hypothesized that A(2A)R overexpression could protect the heart from adriamycin-induced cardiomyopathy. Transgenic (TG) mice overexpressing the A(2A)R and wild-type mice (WT) were injected with adriamycin (5 mg.kg(-1).wk(-1) ip, 4 wk). All WT mice survived adriamycin treatment while A(2A)R TG mice suffered 100% mortality at 4 wk. Telemetry showed progressive prolongation of the QT interval, bradyarrhythmias, heart block, and sudden death in adriamycin-treated A(2A)R TG but not WT mice. Both WT and A(2A)R TG demonstrated similar decreases in heart function at 3 wk after treatment. Adriamycin significantly increased end-diastolic intracellular Ca(2+) concentration in A(2A)R TG but not in WT myocytes (P < 0.05). Compared with WT myocytes, action potential duration increased dramatically in A(2A)R TG myocytes (P < 0.05) after adriamycin treatment. Expression of connexin 43 was decreased in adriamycin treated A(2A)R TG but not WT mice. In sharp contrast, A(2A)R overexpression induced after the completion of adriamycin treatment resulted in no deaths and enhanced cardiac performance compared with WT adriamycin-treated mice. Our results indicate that the timing of A(2A)R activation is critical in terms of exacerbating or protecting adriamycin-induced cardiotoxicity. Our data have direct relevance on the clinical use of adenosine agonists or antagonists in the treatment of patients undergoing adriamycin therapy.

Fig. 1.

A: survival after adriamycin (Ad) treatment. All wild-type (WT) mice survived adriamycin treatment, while adenosine A_2A receptor (A_2AR) transgenic (TG) mice had 100% mortality by the end of 4 wk (P < 0.05, log-rank test, n = 10). DOX, doxycyclin. B: evaluation of cardiac function after third adriamycin treatment in A_2AR TG and WT mice. Adriamycin reduced cardiac function in both WT and A_2ATG mice. Percent fractional shortening (FS) of indicated mouse groups is shown. *P < 0.01 WT baseline vs. WT adriamycin. +P < 0.001 A_2AR TG baseline vs. A_2AR TG adriamycin. NS, not significant WT adriamycin vs. A_2AR TG adriamycin (see Table 1 for details). C: electron microscopic images of myocardial sections of WT (n = 2) and A_2ATG (n = 2) mouse hearts after third adriamycin injection. m, Mitochondrion. Bar = 500 nM.

Fig. 2.

Telemetric tracings from A_2AR TG and WT mice treated with adriamycin. A: telemetric data from A_2AR TG and WT mice (n = 3 for each group). Progressive prolongation of QT segment (ms) with each injection in A_2AR TG (P < 0.001) is shown. WT mice had some prolongation in QT interval after the third injection, but they had no arrhythmias. *P < 0.05 (n = 3 mice for each group, male 10- to 12-wk-old mice). B: development of runs of ventricular tachycardia, bradyarrhythmias, and first- and second-degree heart block.

Fig. 3.

Intracellular Ca²⁺ concentration ([Ca²⁺]_i) transients and action potential durations. After adult myocytes from WT and A_2ATG were isolated, 5 μM adriamycin was added to the culture medium and measurements were made 18 h after exposure. A: representative tracing of myocyte calcium transients in A_2AR TG and WT mice with and without adriamycin. B: representative tracing of action potential duration measurements in A_2AR TG and WT mice with and without adriamycin.

Fig. 4.

Connexin 43 (Cx43) staining patterns and expression in A_2AR TG and WT mice treated with adriamycin. A: after the third dose of adriamycin, ventricular myocardium from A_2AR TG and WT mice was immunostained with Cx43 and β-catenin (n = 3 for each group). B: ventricular extracts were prepared from hearts not exposed to adriamycin (left) and from hearts after 3 injections of adriamycin (right). Ten- to 12-wk-old male WT hearts (n = 4) and A_2A TG hearts (n = 4–6) were probed for Cx43 and dephosphorylated (De-P Cx43, 13-8300). Cx43 signals were normalized to GAPDH. Values are means ± SE. *P < 0.05 vs. WT.

Fig. 5.

Effect of A_2AR expression after cessation of adriamycin treatment. A: schematic diagram showing DOX inhibition and induction of A_2AR in 8-wk-old TG mice. Mice were harvested at indicated times after DOX removal. Total ventricular protein extracts were immunoblotted with anti-A_2AR antibody. B: schematic diagram showing induction of A_2AR expression after cessation of adriamycin administration. Survival curve showed that control mice treated with adriamycin (n = 24) had over 30% mortality, while all A_2AR TG mice (n = 7) survived. C: relative cardiac function in A_2AR TG and control mice 8 wk after adriamycin administration. As detailed in materials and methods, this echocardiographic experiment was performed using the ACUSON Sequoia C256 system and Avertin anesthesia. Exact %FS: WT no adriamycin (53.0 ± 1.4, n = 8), WT post-adriamycin (45.2.0 ± 1.9, n = 12); A_2AR TG post-adriamycin (52.7 ± 1.3, n = 6). *P < 0.05 vs. WT post-adriamycin.