INTRODUCTION

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A NOVEL ADENOSINE A₁/A₂ receptor agonist, {[1S-[1,2, 3,4(S*)]]-4-[7-[[2-(3-chloro-2-thienyl)-1-methylpropyl]- amino]-3H-imidazo[4,5,-b]pyridin-3-yl]cyclopentane carboxamide} (AMP 579), has been reported to protect the ischemic heart in pig (33), dog (20), and rabbit (6, 40) models by limiting infarct size. Most importantly, the protective effect of AMP 579 in the above studies was seen when it was administered at the time of reperfusion. The protection of AMP 579 requires adenosine receptor activation, yet adenosine cannot duplicate its effect (6, 40). We (39) have demonstrated that AMP 579 profoundly reduces postischemic contracture, implying a beneficial effect on intracellular calcium homeostasis at reperfusion. However, the ultimate mechanism of protection of AMP 579 is still unknown. Because AMP 579 is so effective when given at reperfusion, it is reasonable to propose that it blocks reperfusion injury. Many studies (5, 12, 13, 19) suggest that reperfusion injury is the result of reactive oxygen species (ROS) generated within the reperfused heart. Therefore, it is possible that AMP 579 prevents myocardial reperfusion injury, at least in part, by reducing oxidant stress. The objective of this study was to observe whether AMP 579 could modulate oxidant stress in cultured embryonic chicken cardiomyocytes subjected to simulated ischemia and reoxygenation.

	MATERIALS AND METHODS

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Cardiac cell culture. Ten-day-old embryonic chick ventricular myocytes were prepared using a method first described by Barry et al. (2) and modified by Vanden Hoek et al. (37). Briefly, hearts were harvested and placed in Hanks' balanced salt solution lacking magnesium and calcium. Ventricles were minced, and myocytes were dissociated by treatment with trypsin (0.025%, Life Technologies; Grand Island, NY) applied four to six times at 37°C with gentle agitation. Isolated cells were then transferred to a solution with trypsin inhibitor for 8 min, filtered through a 100-µm mesh, centrifuged for 5 min at 1,200 rpm at 4°C, and finally resuspended in a nutritive medium described previously by Chandel et al. (7) and Duranteau et al. (11). Resuspended cells were placed in a petri dish in a humidified incubator (5% CO₂-95% air, 37°C) for 45 min to promote early adherence of fibroblasts. Nonadherent cells were counted with a hemocytometer, and viability was measured using trypan blue (0.4%). Approximately 1 × 10⁶ cells in the nutritive medium were pipetted onto coverslips (25 mm) and incubated for 3-4 days, after which synchronous contractions of the monolayer were noted. Experiments were performed on spontaneously contracting cells at 3 or 4 days after isolation.

Perfusion system. Glass coverslips containing spontaneously beating chick myocytes were placed in a stainless steel, 1-ml flow-through chamber (Penn Century; Philadelphia, PA). The chamber was sealed with thin water gaskets to minimize oxygen exchange between the chamber wall and the perfusate and was then mounted on a temperature-controlled platform (at 37°C) of an inverted microscope. A water-jacketed glass column mounted above the microscope stage was used to equilibrate the perfusate to a known PO₂. The standard perfusion medium consisted of a buffered salt solution (BSS) containing (in mM) 177 NaCl, 4.0 KCl, 18 NaHCO₃, 0.8 MgSO₄, 1.0 NaH₂PO₄, 1.21 CaCl₂, and 5.6 glucose, which was equilibrated for 1 h before the experiment by bubbling with a gas mixture of 21% O₂-5% CO₂-74% N₂. A simulated ischemia solution, composed of BSS containing 2-deoxy-D-glucose (20 mM) in lieu of glucose to inhibit glycolysis, was bubbled with 20% CO₂-80% N₂ for 1 h before the experiments. The pH of the perfusion solution was routinely measured (normoxic BSS: pH 7.4, simulated ischemic BSS: pH 6.8). Stainless steel or low oxygen solubility polymer tubing connected the equilibration column to the flow-through chamber to minimize ambient oxygen transfer into the perfusate. In previous studies (18, 38), the low PO₂ in the chamber was confirmed using an optical phosphorescence-quenching method (Oxyspot, Medical Systems; Greenvale, NY) under conditions identical to those of these experiments.

Viability assay. The inverted microscope, equipped for epifluorescent illumination, included a xenon light source (75 W), a 12-bit digital cooled charge-coupled device camera (CCD, Princeton Instruments), a shutter, a Sutter filter wheel, and appropriate excitation and emission filter cubes. The microscope was also equipped with Hoffman-modified phase illumination to accentuate surface topology. Fluorescent cell images were obtained using a ×10 objective microscope (Nikon Plan Fluor). Data were acquired and analyzed with Metamorph software (Universal Imaging). Cell viability was quantified with the nuclear stain propidium iodide (PI; 5 µM) (Molecular Probes; Eugene, OR), an exclusion fluorescent dye that binds to chromatin on loss of membrane integrity (4, 35, 37). There were ~600 cardiomyocytes under the selected field for each experiment. One representative field of synchronously contracting cells was chosen and monitored throughout the experiments. PI has not been found to be toxic to cells over a course of up to 8 h and therefore was present in the perfusate throughout the experiments. At the completion of each experiment, 300 µM digitonin was added to the perfusate for 1 h. Digitonin disrupted the cell membrane integrity of all cells and allowed PI to enter. Percent loss of viability (cell death) was then calculated by expressing the fluorescence at any time point as a percentage of the fluorescence after 1 h of digitonin exposure (100%). In control experiments with cells oxygenated for 6-8 h, cell death is <2%.

Measurement of ROS generation. ROS generation in cells was assessed using the nonfluorescent probe 2',7'-dichlorofluorescin (DCFH). The membrane-permeable diacetate form of the DCFH dye (DCFH-DA) was added to the perfusate at a final concentration of 5 µM. Within the cell, esterases cleave the acetate groups on DCFH-DA, thus trapping the probe DCFH intracellularly (29). ROS in the cells lead to the oxidation of DCFH, yielding the fluorescent product 2'-7'-dichlorofluorescein (DCF) (29, 30). DCFH in cardiomyocytes is readily oxidized by H₂O₂ or the hydroxyl radical but is relatively insensitive to superoxide (11, 35, 37). Fluorescence was measured using an excitation wavelength of 480 nm, a dichroic 505-nm long-pass mirror, and an emitter bandpass filter of 535 nm (Chroma Technology) with neutral density filters to attenuate the excitation light intensity. Fluorescence intensity was assessed in clusters of several cells identified as regions of interest. The background was identified as an area without cells or with minimal cellular fluorescence. Intensity values are reported as the percentages of initial values after the background value was subtracted.

Experimental protocol. After an equilibration period of 60 min, control cells were subjected to 60 min of simulated ischemia, as detailed above, followed by either 15 min (ROS generation studies) or 3 h (cell viability studies) of reoxygenation. In treatment groups, the cells were exposed to either AMP 579 or adenosine starting 10 min before reoxygenation. To determine which adenosine receptor subtype was responsible for the effect of AMP 579 on ROS production, cells were exposed simultaneously to AMP 579 and 8-(13-chlorostyryl) caffeine (CSC) (1 µM), a selective A₂ antagonist.

Chemicals. AMP 579 was a gift from Aventis Pharmaceuticals. Adenosine, CSC, and all other chemicals were purchased from Sigma.

Statistical analysis. Data are expressed as means ± SE. Two-way analysis of variance with repeated measures and Fisher's test were used to test for differences among the groups. A P value <0.05 was considered to be significant.

	RESULTS

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ROS generation. Figure 1 shows the time course of changes in mean DCF fluorescence intensity for the four study groups. On reoxygenation, the DCF fluorescence intensity in the control group (n = 6) increased dramatically, indicating an increased ROS production after reintroduction of oxygen. AMP 579 at 0.5 µM (n = 6) greatly attenuated the burst of ROS at reoxygenation and production was further attenuated by 1 µM AMP 579 (n = 7). In contrast, adenosine (100 µM) had no effect on the increase in DCF fluorescence. Figure 2 summarizes the peak values of DCF fluorescence during reoxygenation. AMP 579 significantly reduced the peak value from 4.86 ± 0.30 (arbitrary units) in control cells to 2.72 ± 0.31 and 1.85 ± 0.14 in cardiomyocytes exposed to 0.5 and 1 µM AMP 579, respectively (P < 0.05). Adenosine at a concentration of 100 µM (n = 4) did not affect the peak value (5.28 ± 0.22).

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Plots documenting the effect of AMP 579 and adenosine (Ado) in isolated chick cardiomyocytes exposed to simulated ischemia and reperfusion (reperf) on fluorescence of 2'-7'-dichlorofluorescein (DCF), expressed in arbitrary units (A.U.), an index of generation of reactive oxygen species (ROS).

View larger version (50K): [in this window] [in a new window]   Fig. 2.   Peak values of DCF fluorescence representing generation of ROS in isolated chick cardiomyocytes exposed to AMP 579, adenosine, and 8-(13-chlorostyryl) caffeine (CSC). *P < 0.05 vs control.

Cell viability. Percent cell death (PI uptake) was monitored continuously throughout the experiment and the data are shown in Fig. 3. Reoxygenation was accompanied by the onset of cell death. After 3 h of reoxygenation, cell death was 49.6 ± 4.7% in the control group (n = 8) and was significantly reduced by 1 µM AMP 579 (n = 6) (25.4 ± 2.4%, P < 0.05). In contrast, cell death was not significantly altered by 100 µM adenosine (n = 6) (46.4 ± 2.2%).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effects of AMP 579 and adenosine on cell death. Propidium iodide (PI) was used to provide an index of cell death. *P < 0.05 vs control.

	DISCUSSION

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The present study reveals that AMP 579 can significantly attenuate ROS generation by hypoxic cardiac cells when they are reoxygenated. This attenuation of oxidant stress may well be related to the mechanism by which AMP 579 protects the reperfused heart against infarction. Although an increasing body of data has demonstrated the cardioprotective effects of AMP 579 in various animal models (6, 20, 33, 40), the mechanism underlying the protection of the agent is still unclear. Most baffling is the observation that activation of the adenosine A₂ receptor seems to be a requisite for the protection, yet adenosine or A₂ receptor agonists will not duplicate the protective effect (6, 23).

Nakamura et al. (23) reported that AMP 579 inhibits neutrophil activation and neutrophil-mediated coronary dysfunction, suggesting neutrophil suppression in the action of AMP 579. However, we found that AMP 579 protects against infarction, even in neutrophil-free isolated hearts (40). The present study extends that observation. Cardiomyocyte viability was preserved by the use of AMP 579 in a cell model, which contained no neutrophils or any other cell type, indicating that the cardiomyocyte is the direct target for this drug.

Myocardial reperfusion injury refers to a toxic event caused by the act of reperfusion. If a reperfusion injury is present, it should be possible to attenuate it by an intervention given at the time of reperfusion (27). There are several hypotheses regarding the mechanism by which reperfusion injury might occur in the heart. These include calcium overload (24), apoptosis (1), osmotic swelling (15), and the generation of ROS. Whereas it is clear that reperfusion of the ischemic heart is accompanied by a burst of ROS, the consequence of their presence is controversial. In a previous study (36) of chick cardiomyocytes, both the free radical scavenger N-2-mercaptopropionyl glycine and the ferrous ion chelator 1,10-phenanthroline attenuated ROS production while increasing cell viability and improving return of cell contraction. There are compelling data to indicate that ROS generated at reperfusion contribute to stunning of myocardium (3). Stunned myocardium is a temporary lesion in which the contractility of the heart is greatly reduced. However, unlike infarction, these effects are fully reversible. It has been proposed that ROS contribute to infarction of the heart as well. The evidence supporting a contribution to the infarction process was gathered from reports that treatment with antioxidants given only at reperfusion could reduce infarct size in animal models (14, 16, 22). Unfortunately, many laboratories have been unable to reproduce those findings (21, 26, 31, 34). As a result, many believe that ROS stun the reperfused heart, but that insufficient ROS are produced to significantly contribute to cell killing (10, 17, 28).

In the present study, AMP 579 greatly attenuated the burst of ROS accompanying reoxygenation. Because the effective concentration of AMP 579 was as low as 0.5 µM, it is unlikely that AMP 579 acted as a direct scavenger, nor could AMP 579 have caused expression of endogenous antioxidants so quickly. The most probable explanation is that AMP 579 exerted a direct effect on myocardial cells, which caused them to generate fewer ROS. The important question then becomes whether suppression of ROS is the mechanism whereby AMP 579 limits infarct size in the whole heart. That could be the case only if ROS actually contribute to infarction. It is also equally possible that AMP 579 acted to preserve the integrity of the cell through some ROS-independent process, and, as a result, the less-injured cells simply produced fewer ROS at reperfusion.

Reperfused hearts are thought to produce ROS at reperfusion as a result of defects in their metabolic pathways. These include defects in electron transport or perhaps activation of Ca²⁺-dependent oxidases as Ca²⁺ enters the injured cell. In a previous study (39), AMP 579 markedly reduced the degree of myocardial contracture on reperfusion in the isolated rabbit heart model, suggesting that the drug acted to keep calcium out of the cells. Oxidant stress could cause a depression in the membrane Ca²⁺ exchangers or the Na⁺-K⁺ ATPase that maintains the sodium gradient for the exchanger (8, 9). Oxidant stress also depresses the sarcoplasmic reticulum Ca²⁺ pump ATPase (25, 32). These changes would result in intracellular Ca²⁺ overload and myocardial contracture. On the other hand, it is just as plausible that AMP kept Ca²⁺ out of the cell by some mechanism unrelated to ROS and, in the absence of elevated Ca²⁺, fewer ROS were produced. In the latter scenario, the reduced ROS would have been the result of the protection rather than its cause. Furthermore, these studies cannot completely exclude the possibility that the effect of AMP 579 on ROS is unrelated to its necrosis-sparing action.

In previous studies, we (40) and others (33) found that the effect of AMP 579 required adenosine receptor activation. The present investigation confirms and extends these earlier observations. A selective adenosine A₂ antagonist completely prevented the ability of AMP 579 to attenuate the burst of ROS upon reperfusion. Thus, somehow, A₂ activity is necessary for AMP 579 to exert its salutary effect. We also evaluated the effect of 100 µM adenosine given with the same timing as AMP 579. That level of adenosine should have nearly saturated both A₁ and A₂ receptors. Yet surprisingly, adenosine was unable to reduce either ROS generation or cell death in these cells. It is unknown how A₂ receptor stimulation participates to produce protection. It appears to be necessary, but not sufficient. These observations suggest that the AMP 579 molecule may possess protective capabilities beyond those of adenosine alone. The fact that both ROS production and cell death responded in parallel fashion to both AMP 579 and adenosine would suggest that the two processes are related to one another.

These studies were performed in cultured embryonic ventricular myocytes. This model has proved to be very effective for the study of ROS production during and after ischemia (35, 37) and for investigation of mechanisms of ischemic preconditioning (35, 41). And it is now helping to uncover the mode of action of a drug known to be quite protective in intact hearts. These cultured cells have surface receptors, can be preconditioned, and contract, and, therefore, are differentiated. One cannot exclude the possibility that some genes are active that are subsequently downregulated in adult cardiomyocytes. But the ability to grow cells into a monolayer to have a fixed, adherent cell population to observe greatly facilitates studies such as those described here.

In summary, the novel adenosine A₁/A₂ receptor agonist AMP 579 given just before simulated reperfusion strongly attenuates the rate at which isolated chick cardiomyocytes die following a period of simulated ischemia. AMP 579 also decreases the burst of ROS seen at reoxygenation suggesting a cause-and-effect relationship between cell death and ROS generation. This effect on ROS production is dependent on adenosine A₂ receptor binding. Yet adenosine could not duplicate either of these effects, indicating that AMP 579 has protective actions apart from its effect on adenosine receptors.