Effects of cardiac-restricted overexpression of the A_2A adenosine receptor on adriamycin-induced cardiotoxicity

Abstract

Activation of the A_2A adenosine receptor (A_2AR) has been shown to be cardioprotective. We hypothesized that A_2AR overexpression could protect the heart from adriamycin-induced cardiomyopathy. Transgenic (TG) mice overexpressing the A_2AR 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_2AR 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_2AR TG but not WT mice. Both WT and A_2AR TG demonstrated similar decreases in heart function at 3 wk after treatment. Adriamycin significantly increased end-diastolic intracellular Ca²⁺ concentration in A_2AR TG but not in WT myocytes (P < 0.05). Compared with WT myocytes, action potential duration increased dramatically in A_2AR TG myoctyes (P < 0.05) after adriamycin treatment. Expression of connexin 43 was decreased in adriamycin treated A_2AR TG but not WT mice. In sharp contrast, A_2AR 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_2AR 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.

anthracycline antibiotics, such as adriamycin (doxorubicin), are antitumor agents that have been used since the late 1960s for treatment of hematological and solid tumor malignancies (44). Despite their efficacy as anticancer agents, they are associated with significant cardiotoxicity. These deleterious effects are dose dependent and may persist long after the treatment is stopped (36). The effects of adriamycin on the heart have been extensively studied and serve as a model for understanding anthracycline-associated cardiomyopathies (18). Adriamycin causes a decrease in fractional shortening (FS) that is accompanied by myofibril loss and vacuolization (17, 46). Adriamycin-associated arrhythmias have also been documented in case reports and include nonspecific T wave and ST changes and rarely sudden death (8, 20). The mechanisms by which adriamycin induces cardiotoxicity have been proposed to include formation of free radicals (19), inhibition of nucleic acid and protein synthesis (4, 45), lipid peroxidation (29, 35, 39), abnormalities in mitochondria (11), lysosomal changes (39), modifications of sarcolemmal Ca²⁺ transport, diminished activity of adenylate cyclase, Na⁺-K⁺-ATPase, and Ca²⁺-ATPase activities (11, 35, 37, 38), and the release of histamines and catecholamines (3, 22).

Adenosine, an ubiquitous purine nucleotide, is cardioprotective during ischemic pre- and postconditioning (15, 21, 51). The biological actions of adenosine are mediated by a family of G protein-coupled receptors found on the sarcolemmal surface of cardiac myocytes, including the A₁, A_2A, and A₃ adenosine receptors (R) and the A_2BR, which is expressed only in the cardiac vasculature. Activation of the A₁R or A₃R pathways inhibits adenylyl cyclase activity through inhibitory guanine nucleotide binding protein αGi, while activation of A_2AR enhances cAMP production through αGs (6, 15, 42). Activation of the A_2AR has been associated with inhibition of the inflammatory response (45, 49). Overexpression of A_2AR resulted in enhanced contractility, increased sarcoplasmic reticulum (SR) Ca²⁺ uptake, and a higher systolic Ca²⁺ concentration. Therefore, we hypothesize that A_2AR overexpression might be protective in adriamycin-induced cardiomyopathy. To test this hypothesis, we induced overexpression of A_2AR in transgenic mice (TG), either concomitant with or after adriamycin administration, and compared cardiac performance and survival with adriamycin-treated wild-type (WT) animals.

MATERIALS AND METHODS

TG mouse generation.

Mice with inducible, cardiac-specific overexpression of the human A_2AR (A_2A TG) were engineered on a FVB background as previously described (5, 9). A_2A TG mice were crossed with mice that expressed tetracycline transactivator (tTA) in the heart (tTA TG) in the presence of doxycycline (Dox). In this “tet-off”-inducible system, Dox, the stable tetracycline analog, inhibits tTA transactivation and was administered to mice at 300 mg/kg of a mouse diet (Bio-Serv). Dox was removed from mice to induce transgene expression. Mice were housed and fed on a 12:12-h light-dark cycle at the Thomas Jefferson University Animal Facility and were supervised by veterinary staff members. Standard care was provided to all mice used for experiments. All protocols applied to the mice in this study were approved and supervised by the Institutional Animal Care and Use Committee of Thomas Jefferson University.

Chronic mouse model of adriamycin-induced cardiotoxicity.

In initial experiments, Dox was removed from the animals' diets at 3 wk of age to assess the concomitant effects of adriamycin administration and activation of the A_2AR pathways. To this end, 8- to 12-wk-old male A_2AR TG mice and WT FVB controls (10 mice/group) were injected with adriamycin (5 mg·kg⁻¹·wk⁻¹ ip for 4 wk; Sigma-Aldrich, St. Louis, MO). This regimen was designed to simulate an adriamycin dose of 1,400 mg/70 kg which is associated with development of cardiomyopathy in humans (36). It should be noted that since adriamycin-treated A_2AR TG mice had a low survival rate (Fig. 1), measurements of gene and protein expression and cardiac function were made in mice that were killed earlier in the cycle of adriamycin therapy (1 day after the third adriamycin injection when most of treated A_2A TG mice were alive). In a second series of experiments, the effects of A_2AR activation were assessed after adriamycin treatment was complete. Eight-week-old A_2AR TG mice (n = 7) and WT mice (n = 24) were maintained on Dox (since birth) and treated with adriamycin (5 mg·kg⁻¹·wk⁻¹ ip) for 4 wk. Dox was withdrawn after completion of adriamycin treatment, and the animals were monitored for an additional 8 wk.

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.

In vivo assessment of cardiac function.

Left ventricular (LV) function in mice was evaluated with transthoracic two-dimensional echocardiography (TTE). In experiments depicted in Fig. 1 and Table 1, mice were anesthetized with 2% inhaled isofluorane, and LV function at baseline and at 1 day after the third injection of adriamycin was assessed with the VisualSonics VeVo 770 imaging system with a 707 scanhead. This experiment assessed LV function at baseline and at 1 day after the third injection of adriamycin. In experiments depicted in Fig. 5 in which LV function was evaluated 8 wk after cessation of adriamycin treatment, mice were anesthetized with 2.5% Avertin (10 μl/g body wt ip, Aldrich Chemical) and echocardiographic studies were performed using the ACUSON Sequoia C256 system (5). Age-matched, non-TG or tTA mice on a FVB background served as controls. TTE in M-mode was carried out in the parasternal short-axis view at the papillary muscle level to assess LV end-diastolic diameter (LVEDD) and LV end-systolic diameter (LVESD). FS was calculated as %FS = [(LVEDD − LVESD)/LVEDD] × 100 (30).

Table 1.

Echocardiography of WT and A_2AR TG mice before and after adriamycin injection

	WT (5)	WT Ad (5)	A2AR TG (7)	A2AR TG Ad (6)
Heart rate, bpm	410 ± 42	476 ± 12	557 ± 11	498 ± 18
LVEDD, mm	3.4 ± 0.2	3.5 ± 0.1	3.3 ± 0.1	3.6 ± 0.1
LVESD, mm	2.0 ± 0.2	2.35 ± 0.2	1.8. ± 0.1	2.5 ± 0.1
%FS	40.1 ± 2.1	33.1 ± 2.6*	45.2 ± 2.7	32.1 ± 2.3*

Values are means ± SE. Numbers in parentheses are number of mice per group (8- to 12-wk male mice). WT, wild-type; WT Ad, WT after 3rd injection of adriamycin (Ad); A_2AR TG, adenosine A_2A receptor-overexpressing transgenic mice; A_2AR TG Ad, A_2AR TG mice after 3rd injection of adriamycin; LVEDD and LVESD, left ventricular end-diastolic and -systolic dimensions, respectively; %FS, percentage of fractional shortening. This experiment used mice anesthetized by 2% inhaled isoflurane and the VisualSONICS VeVo 770 imaging system with a 707 scanhead.

^*P < 0.05 compared with same mouse type without adriamycin.

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.

Electron microscopy.

WT and A_2AR male 8- to 12-wk-old mice (n = 2 for each group) were injected with doxorubicin. After the third injection, hearts were perfused with NaCl solution (0.8%) and then with fixative (4% paraformaldehyde, 2% gluteraldehyde in 0.1 M cacodylate buffer) and harvested. Portions of the LV were cut into 1-mm² cubes and washed three times in cacodylate buffer followed by dehydration through graded alcohols and propylene oxide. The samples were embedded in EM bed 812 (Electron Microscopy Sciences, Hatfield, PA). Longitudinal and transverse sections were cut on an UltraCut E ultramicrotome and stained with uranyl acetate and lead citrate (Electron Microscopy Sciences). Images were collected with an AMT XR41-B 4 megapixel camera on a Hitachi H-7000 electron microscope.

Isolation of adult murine cardiac myocytes.

Cardiac myocytes were isolated from the septum and LV free wall of WT and A_2AR TG mice (male, 8–12 wk old) as previously described (32). Briefly, mice were heparinized (1,500 U/kg ip) and anesthetized (pentobarbital sodium, 50 mg/kg ip). Excised hearts were mounted on a steel cannula and retrograde perfused (100 cmH₂O, 37°C) with Ca²⁺-free bicarbonate buffer followed by enzymatic digestion (collagenases B and D, protease XIV). Isolated myocytes were cultured on laminin-coated glass coverslips, and the Ca²⁺ concentration of the buffer was incrementally increased from 0.05 to 0.5 mM (0.05, 0.125, 0.25, 0.5 mM) with 10 min of exposure at each Ca²⁺ concentration. The 0.5 mM Ca²⁺ buffer was then aspirated and replaced with MEM (Sigma-Aldrich) containing 1.2 mM Ca²⁺, 2.5% FBS, and antibiotics (1% penicillin/streptomycin). After 1 h (4% CO₂, 37°C), media were replaced with FBS-free MEM. Preliminary studies were performed to establish the appropriate adriamycin dose to use in single-cell studies. Adriamycin at 5 μM induced lethality in ∼50% of myocytes at 24 h (data not shown). Based on these studies, myocytes from A_2AR TG and WT mice were treated with 5 μM adriamycin for 18 h (n = 3 mice for each group).

Intracellular Ca²⁺ concentration transient measurements.

Myocytes from A_2AR TG and WT mice pretreated with adriamycin or vehicle were exposed to 0.67 μM fura 2-AM for 15 min at 37°C. Fura 2-loaded myocytes were field-stimulated to contract (1 Hz, 37°C) in medium 199 containing 1.8 mM extracellular Ca²⁺ concentration ([Ca²⁺ ]_o). Intracellular Ca²⁺ concentration ([Ca²⁺]_i) transient measurements were performed as previously described (28).

Action potential measurements.

Action potentials were recorded using a current-clamp configuration at 1.5× threshold stimulus and 4-ms duration (47, 54). The pipette solution consisted of (in mM) 125 KCl, 4 MgCl₂, 0.06 CaCl₂, 10 HEPES, 5 K⁺-EGTA, 3 Na₂ATP, and 5 Na₂-creatine phosphate (pH 7.2). The external solution consisted of (in mM) 132 NaCl, 5.4 KCl, 1.8 CaCl₂, 1.8 MgCl₂, 0.6 NaH₂PO₄, 7.5 HEPES, 7.5 Na⁺-HEPES, and 5 glucose, pH 7.4.

Immunofluorescence analysis of connexin 43 and β-catenin.

Hearts were isolated from WT and A_2AR TG mice after the third weekly dose of adriamycin. Indirect immunofluorescence was performed on paraffin-embedded sections of hearts as previously described (7). The sections were incubated with antibodies against β-catenin and connexin 43 (Cx43; Zymed Labs, South San Francisco, CA) overnight at 4°C. Secondary antibodies were Alexa Fluor 488-conjugated goat anti-rabbit IgG (Molecular Probes, Carlsbad, CA) for 1 h. The double-stained sections were imaged with a Zeiss Meta confocal microscope (Thornwood, NY).

Immunoblotting.

Frozen ventricular tissues were homogenized on ice using a nonionic detergent-based lysis buffer containing (in mM) 25 Tris·HCl (pH 7.6), 137 NaCl, 10% glycerol, 1% NP-40 or IGEPAL CA-630, and 10 NaF and freshly supplemented with (in mM) 1 sodium pyrophosphate, 1 EDTA, 10 PMSF, and 1 NaVO₄ and leupeptin, and aprotinin (5 μg/ml each). After separation on 4–12% SDS-PAGE, fractionated proteins were transferred onto nitrocellulose membranes. Both total Cx43 and dephosphorylated Cx43 (mouse monoclonal antibodies that bind selectively to S364/S365; kind gift from Dr. P. D. Lampe, Fred Hutchinson Cancer Research Center, Seattle, WA) were measured as previously described (24). Signals were normalized to GAPDH signals on the same blot. Blots were incubated with either IRDye 700 or 800 secondary antibodies and visualized using Odyssey Infrared Imaging System software (Li-Cor, Lincoln, NE).

Telemetry device implantation, data acquisition and processing.

To assess the cause of sudden death, DSI transmitters (20 × 10 × 8 mm, TA10ETA-F20; Data Sciences International, St. Paul, MN) were chronically implanted in WT and A_2AR TG mice (n = 3 for each group). This transmitter, together with platform receivers (32 × 22 × 3 cm, RPC-1), permit high-quality ECG recordings during sustained physical activity (34). Briefly, the body of the transmitter was placed in the abdominal cavity of anesthetized (10 μl/g Avertin) mice, and the two electrodes were fixed to the dorsal surface of the xiphoid process and the anterior mediastinum close to the right atrium. Ibuprofen (1 ml/100 ml of water) was used for analgesia for 24–48 h. Mice were allowed to recover for 2 wk before adriamycin administration. Continuous ECG recordings were performed at baseline and 24–48 h after each weekly adriamycin injection. Data acquisition was performed with the Data ART acquisition system (Data Sciences International). Raw ECG waveforms were scanned for arrhythmias by two independent observers and processed with the Ponemah Analysis Program Version 4.9. (Data Sciences International). The following ECG parameters were quantified: 1) mean R-R, QT, QTc, QRS, ST, PR intervals, R height, and T height duration; and 2) incidence of ventricular and supraventricular arrhythmias.

Statistics.

All results are expressed as means ± SE. Kaplan-Meier survival curves were compared between groups using log-rank tests. One-way analysis of variance was used to analyze [Ca²⁺]_i transients and action potential parameters. Mann-Whitney nonparametric methods were used to compare between two groups. A commercial software package was used for all statistical analysis (Graph Pad Software, La Jolla, CA). A P value of <0.05 was considered statistically significant.

RESULTS

Increased mortality in A_2AR TG mice after treatment with adriamycin.

When A_2AR overexpression was induced 5 wk before adriamycin treatment, A_2AR-overexpressed mice suffered 100% mortality 1 wk after the fourth adriamycin injection while all WT mice survived adriamycin treatment (P < 0.05, Fig. 1A). In a separate cohort, cardiac function was evaluated at baseline and 1 day after the third injection of adriamycin (when >50% of A_2A TG mice were still alive). Baseline %FS was significantly higher in A_2A TG compared with WT mice (Fig. 1B, Table 1). After three injections of adriamycin, both WT and A_2A TG mice demonstrated significantly reduced FS (Fig. 1B, Table 1). The heart weight/body weight ratio was not different in the two groups (data not shown). Electron microscopy showed a striking noninflammatory myopathy (sarcomere loss, disarray of myofibrils, and increase in the number of mitochondria) consistent with adriamycin cardiotoxicity in both WT and A_2AR TG mice exposed to adriamycin (Fig. 1C). Telemetry demonstrated progressive prolongation of the QT interval, runs of ventricular tachycardia, bradyarrhythmias, and first- and second-degree heart block in A_2AR TG mice treated with adriamycin (Fig. 2). Asystole and sudden death were also recorded in A_2AR TG mice and was preceded by runs of nonsustained ventricular tachycardia (Fig. 2B). By contrast, WT mice treated with adriamycin did not demonstrate any arrhythmias.

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.

Effects of adriamycin on [Ca²⁺]_i transients in A_2AR TG and WT mice.

To explore cellular mechanisms responsible for sudden death in A_2AR-overexpressed mice treated with adriamycin, we measured [Ca²⁺]_i transients in isolated WT and A_2AR TG myocytes after exposure to vehicle or 5 μM adriamycin for 18 h. Preliminary studies showed that at this dose, adriamycin-induced lethality was similar between WT and A_2A TG myocytes (data not shown). In the absence of adriamycin, myocytes from A_2AR TG mice had significantly higher (P < 0.001) systolic [Ca²⁺]_i compared with WT myocytes (Table 2, Fig. 3A). Adriamycin caused a significant prolongation in t_1/2 of [Ca²⁺]_i transient decline in both WT and A_2AR TG myocytes. However, adriamycin induced higher end-diastolic [Ca²⁺]_i in the A_2AR TG group compared with the WT group (Table 2).

Table 2.

Effects of adriamycin on [Ca²⁺]_i transients and SR Ca²⁺ uptake

[Ca2+]o	WT (18)	WT Ad (13)	A2AR TG (20)	A2AR TG Ad (18)
Systolic [Ca2+]i, nM	202.3 ± 8.6	210.1 ± 20.6†	308.5 ± 20.7*	318.5 ± 19.7*‡
Diastolic [Ca2+]i, nM	88.3 ± 3.9	105.8 ± 9.7*	88.3 ± 2.7	132.54 ± 9*†‡
[Ca2+]i transient amplitude, % increase in fura 2 signal	19.7 ± 1.0	16.03 ± 1.7*†	33.7 ± 2.3*	23.8 ± 1.7†‡
t1/2 of [Ca2+]i transient decline, ms	142.8 ± 8 (16)	203 ± 20*† (7)	134.4 ± 13 (8)	176.8 ± 21 (5)

Values are means ± SE. For calcium transient measurements, numbers in parentheses are myocytes pooled from 3 mice/treatment group. [Ca²⁺]_i and [Ca²⁺]_o, intracellular and extracellular Ca²⁺ concentration, respectfully.

^*P < 0.05 compared with WT.

^†P < 0.05 compared to A_2AR TG.

^‡P < 0.05 WT-Ad vs. A_2AR TG Ad.

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.

Adriamycin depolarized membrane potential and prolonged action potential in A_2AR TG but not in WT mice.

Another cellular mechanism that may explain sudden death in A_2AR TG mice exposed to adriamycin is prolongation of the action potential. Treatment with adriamycin depolarized membrane potential in A_2AR TG but not WT myocytes (Table 3). At baseline, A_2AR TG myocytes exhibited slightly prolonged action potential duration at both 50 (APD₅₀) and 90% repolarization (APD₉₀) compared with WT myocytes (P < 0.05) (Table 3, Fig. 3B). In addition, exposure to adriamycin resulted in dramatic prolongation in APD₉₀ in A_2AR TG but not in WT myocytes (P < 0.005; Table 3).

Table 3.

Effects Adriamycin on action potential in A_2AR TG and WT mice

	WT (7)	WT Ad (4)	A2AR TG (12)	A2AR TG Ad (13)
Resting Em, mV	−65.2 ± 2.7	−68.7 ± 2.2	−66.7 ± 2.2	−57.89 ± 1.8†‡
AP amplitude, mV	110.3 ± 6.4	106.6 ± 5.7	106.3 ± 3.9	103.12 ± 4.98
APD50, ms	4.9 ± 0.71	5.6 ± 0.45	9.9 ± 1.8*	10.22 ± 1.45*
APD90, ms	25 ± 3.6	25.2 ± 4.5	43.8 ± 3.8*	64.89 ± 5.57*†‡

Values are means ± SE. For action potential measurements, numbers in parentheses are myocytes pooled from 3 mice/treatment group. E_m, membrane potential; AP, action potential; APD₅₀ and APD₉₀, action potential duration at 50 and 90% repolarization, respectively. Cells were paced at 1 Hz.

^*P < 0.05 compared with WT.

^†P < 0.05 compared to A_2AR TG.

^‡P < 0.05 WT-Ad vs. A_2AR TG Ad.

Adriamycin decreased Cx43 and N-cadherin in A_2AR TG but not in WT mice.

Cx43 is the predominant gap junction protein expressed in the myocardium. Loss of the gap junction protein Cx43 has been shown to slow myocardial conduction velocity and induce unidirectional block, resulting in an arrhythmogenic substrate and sudden cardiac death in mice (12–14). After three doses of adriamycin treatment, immunofluorescence imaging showed sparse Cx43 signals at A_2AR TG myocyte junctions compared with WT controls (Fig. 4A). Immunoblotting Cx43 from ventricular extracts not exposed to adriamycin and from posttreatment extracts showed that Cx43 protein expression was significantly less (P < 0.05) in hearts overexpressing A_2AR than in those from WT controls (Fig. 4B). This decrease was associated with a decrease in Cx43 phosphorylation.

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.

A_2AR overexpression after adriamycin treatment improved cardiac performance and survival.

Our data thus far indicated that A_2AR overexpression concomitant with adriamycin administration not only did not afford protection but was actually detrimental. Another clinically relevant protective strategy is to activate A_2AR signaling after adriamycin treatment is complete. When Dox was removed after completion of adriamycin treatment, A_2AR expression was induced in a time-dependent manner (Fig. 5A). In contrast to increased mortality associated with simultaneous activation of A_2AR and adriamycin treatment (Fig. 1A), induction of A_2AR overexpression after cessation of adriamycin administration enhanced survival (Fig. 5B) and improved cardiac function compared with the WT adriamycin-treated controls (Fig. 5C).

DISCUSSION

Adriamycin is known to cause a dose-dependent cardiomyopathy (36) with a decrease in FS associated with myofibril loss and vacuolization in the myocardium (17, 46). In agreement, our current results demonstrated noninflammatory myopathy with decreased FS in both WT and A_2AR TG mice treated with adriamycin. Oxidative damage has been shown to contribute to the toxic effects of adriamycin in mitochondria, the SR, and myofibrils. Damage to cardiomyocytes contributes to remodeling of the ventricle with a globular geometry that can increase wall stress. In addition, adriamycin has been shown to decrease SR Ca²⁺ uptake activity and enhance Ca²⁺ release by the ryanodine receptors in rabbits (33), which can lead to abnormalities in Ca²⁺ homoeostasis and excitation contraction coupling (38). Our current results are in agreement with these earlier studies in that SR Ca²⁺ uptake (as estimated by t_1/2 of [Ca²⁺]_i transient decline) was decreased in both WT and A_2AR TG myocytes exposed to adriamycin. In addition, adriamycin resulted in significantly elevated end-diastolic [Ca²⁺]_i, likely explained by decreased SR Ca²⁺ uptake and enhanced SR Ca²⁺ release by ryanodine receptors. Adriamycin has been associated with a variety of arrhythmias in both animal models and humans (8, 20). However, the mechanisms responsible for these arrhythmias remain undetermined. Many transient nonspecific EKG abnormalities have been described including nonspecific ST T-wave changes, a decrease in QRS voltage, right-axis deviation, T-axis abnormalities, and prolongation of the QT interval. There are a few case reports of heart block, ventricular ectopy, and rare cases of sudden death after adriamycin treatment (8, 20).

Given the known cardioprotective properties of the A_2AR in ischemia, we hypothesized that cardiac-restricted A_2AR overexpression would be beneficial in adriamycin cardiotoxicity. We initially induced A_2AR overexpression concomitant with adriamycin administration. Surprisingly, this maneuver proved to be deleterious rather than cardioprotective, as exposure to adriamycin resulted in sudden death at 4 wk. Thus one of the major findings of our present study is that concomitant activation of A_2AR signaling and adriamycin resulted in progressive prolongation in QT interval, heart block, development of ventricular arrhythmias, and sudden death. Since sudden death is rare after adriamycin treatment in WT animals (5, 16), the deleterious effects of adriamycin observed in the present study were most likely mediated by A_2AR signaling. A second major finding is that when A_2AR overexpression was induced after completion of adriamycin treatment, both survival and cardiac function were better than that seen in the WT controls.

Improper calcium regulation is likely responsible for ventricular arrhythmias seen in mice in which A_2AR overexpression was induced concurrent with adriamycin administration. In the canonical G_s-coupled signaling, the A_2AR activates adenylyl cyclase, generates intracellular cAMP, and activates PKA. Then, PKA activates SERCA2 and elevates systolic [Ca²⁺]_i by phosphorylating both the SERCA2a inhibitor phospholamban (PLN) and the PLN inhibitor complex (PP1/inhibitor 1) (10, 53). At the cellular level, myocytes isolated from A_2AR TG hearts and treated with adriamycin demonstrated significantly higher end-diastolic [Ca²⁺]_i, decreased SR Ca²⁺ uptake as indicated by longer t_1/2 of [Ca²⁺]_i transient decline, depolarized membrane potential, and prolongation of APD₉₀. Elevated diastolic [Ca²⁺]_i promotes forward Na⁺/Ca²⁺ exchange (3 Na⁺ in: 1 Ca²⁺ out), which generates an inward current during diastole, leading to membrane depolarization and afterdepolarizations. Action potential prolongation and afterdepolarizations are commonly incriminated as cellular substrates for arrhythmogenesis. Thus the sudden death seen in mice overexpressing the A_2AR and treated with adriamycin may be explained by development of cardiac arrhythmias caused by altered [Ca²⁺]_i homeostasis.

Another cellular mechanism that may account for increased arrhythmogenesis resulting in sudden death is structural changes in gap junctions (16). In the present study, the finding that adriamycin significantly altered the levels of the junctional protein Cx43 provided an alternative explanation for the marked increase in sudden death. Cx43 mechanically and electrically couples cardiomyoctes to ensure rhythmic contraction. Cardiac-specific loss of Cx43 slows myocardial conduction velocity, which leads to arrhythmias and sudden cardiac death (12–14). Recent studies have also demonstrated that cardiac-specific loss of Cx43 or change in Cx43 localization can induce unidirectional block, resulting in an arrhythmogenic substrate and sudden cardiac death in mice (12–14). Arrhythmic susceptibility in N-cadherin/Cx43 compound heterozygous mice was associated with a reduced Cx43 protein level and an increase in the dephosphorylated Cx43 level (23, 24). Changes in Cx43 phosphorylation regulate Cx43 trafficking, assembly, gating, and turnover in the cell (41), and progressive dephosphorylation of Cx43 is associated with electrical uncoupling (1, 2). Thus the significant decrease in Cx43 and accompanying changes in intercellular coupling may lead to the high incidence of bradyarrhythmias and sudden death observed in A_2AR TG mice exposed to adriamycin. However, levels of Cx43 were significantly reduced in A_2AR mice even in the absence of adriamycin. Therefore, while changes in Cx43 may increase susceptibility to arrhythmia in the presence of adriamycin and A_2AR activation, our present study did not go into sufficient depths to accept or refute this hypothesis.

We cannot exclude the possibility that alterations in K⁺ channel activity or levels might also have contributed to the sudden death seen in A_2AR TG animals exposed to adriamycin; however, this explanation is less likely. Abnormalities in K⁺ channels have been implicated in long QT syndrome, short QT syndrome, and familial atrial fibrillation, all of which lead to tachyarrhythmias rather than bradyarrhythmias or heart block as seen in our A_2AR TG mice treated with adriamycin.

In contrast to the catastrophic survival associated with concurrent A_2AR activation and adriamycin treatment, we found that induced overexpression of A_2AR after adriamycin therapy was completed was beneficial, both in terms of cardiac function and survival. There are clinical precedents to this finding. For example, the coadministration of adriamycin and herceptin results in as much as a 27% incidence of heart failure in women with metastatic breast cancer (40). By contrast, the administration of herceptin after completion of adriamycin therapy substantially lowers the risk (31). The reason for this differential response remains undefined but may be related to the acute ability of adriamycin to induce free radicals, alter oxidation-related genes (48, 52), and elevate diastolic calcium (this paper and Ref. 43). After cessation of adriamycin exposure, oxidation-related gene alterations rapidly return to normal (within 1–3 days).

Although we did not elucidate the exact cellular mechanisms by which induced A_2AR overexpression after cessation of adriamycin therapy afforded cardioprotection, some insights can be gleaned from the known effects of A_2AR activation. A_2AR signaling may ameliorate adriamycin cardiotoxicity by enhancing SERCA2 activity. Analogous to the proposed mechanisms for SERCA2 overexpression in heart failure (10), A_2AR activation may enhance systolic function, reduce hypertrophy, and improve cardiac energy metabolism. Alternatively, A_2ARs could also mediate cardiac protection independently of the canonical Gs/cAMP/PKA/SERCA2 pathway, as A_2AR binds other proteins and forms heteromeric complex with other G protein-coupled receptors (53). Other signaling pathways activated by A_2AR include MAPKs, PKC, and Akt and nitric oxide (27, 50, 53). The G_s-independent mechanism is likely mediated through the unique A_2AR C terminus that binds accessory proteins such as α-actinin, ARNO, USP4, and translin-associated protein-X. It is through ARNO (the guanine nucleotide exchange factor for ARF6 small G proteins) that A_2AR regulates G_s-independent functions such as endocytosis, arrestin interaction, and MAPK activation (53).

In summary, our study demonstrated that the concurrent activation of A_2AR potentiated the cardiotoxic effects of adriamycin. Increased cardiotoxicity was not due to additional reduction in cardiac contractility but was associated with elevated end-diastolic [Ca²⁺]_i, altered [Ca²⁺]_i homeostasis, decreased Cx43 in gap junctions, and prolonged APD with the subsequent development of cardiac arrhythmias and sudden death. By contrast, activation of A_2AR signaling after completion of anthracycline therapy had salutary benefits. Our findings have significant clinical relevance regarding the use of A_2A adenosine agonists or A₁ adenosine antagonists in patients with diminished LV function secondary to anthracyline therapy.

GRANTS

This work was supported in part by National Heart, Lung, and Blood Institute Grants PO1-HL-91799 (Project 2; to A. M. Feldman) and RO1-HL58672 and RO1-HL74854 (to J. Y. Cheung).

DISCLOSURES

No conflicts of interest are declared by the authors.