An irreversible A₁-selective adenosine agonist preconditions rabbit heart

Abstract

This study tested whether an irreversible agonist of the A₁ adenosine receptor, in DITC ADAC, can mimic the protective effect of ischemic preconditioning in the rabbit heart. Isolated Krebs buffer-perfused rabbit hearts experienced 30 mins of regional ischemia and 120 mins of reperfusion. Infarct size was measured with tetrazolium staining. In untreated hearts 32±2% of the risk zone infarcted while only 9±2% infarction was seen in hearts that were preconditioned with 5 mins of global ischemia followed by 10 mins of reperfusion (P<0.05 versus control). Exposure to 200 nM of the A₁- selective agonist 2-chloro-N⁶-cyclopentyladenosine (CCPA) for 5 mins followed by 10 mins of washout protected the hearts as well as preconditioning with 13±7% infarction (P<0.05 versus control). Protection from CCPA was completely blocked by 200 nM DPCPX (8-cyclopentyl-1,3-dipropylxanthine) with 34±7% infarction (P<0.05 versus CCPA) confirming that protection was via the A₁ adenosine receptor. m-DITC ADAC, which irreversibly stimulates the A₁ adenosine receptor, also protected the hearts with only 15±4% infarction (P<0.05 versus control). It was concluded that m-DITC ADAC does mimic ischemic preconditioning and that an irreversible agonist might be a novel way to provide an extended window of protection to the heart from a single intracoronary injection.

There is great interest in identifying interventions that could make the heart more resistant to infarction during an ischemic insult. One approach that has received much attention in recent years is ischemic preconditioning. First described by Murry and colleagues (1), it was shown that exposing the heart to brief periods of ischemia caused minimal injury but, paradoxically, this caused the heart to become very resistant to infarction from a subsequent ischemic insult. It was later shown by Liu et al (2) that adenosine released by the heart during the preconditioning ischemia triggers the effect, because the protection from ischemic preconditioning could be blocked with nonselective adenosine receptor blockers. It was presumed that the A₁ adenosine receptor (A₁AR) was responsible because a 5 min intracoronary infusion of the A₁-selective agonist R(−)N⁶ -(2-phenylisopropyl) adenosine was as effective as either adenosine or 5 mins of preconditioning ischemia in protecting the heart. Agonists of the A₁AR have also been shown to mimic preconditioning in dog (3), in pig (4–6) and in human (7) myocardium. More recent studies suggest that the A₃ adenosine receptor (A₃AR) may also be participating in this response (8,9), and the separate contribution of the A_lAR versus the A₃AR remains to be clarified.

While the protection conferred by ischemic preconditioning is profound, it is also short lived. Various investigators have found that the protection from ischemic preconditioning wanes within 1 h or less (10–12). Furthermore, Yao and Gross (13) report that pharmacological preconditioning with adenosine wanes even faster than that from ischemia. One way to circumvent this problem might be the use of an irreversible agonist for the A_lAR. Such a compound, m-DITC-ADAC (N⁶-[4-[[[4-[[[[2-[[[(3-isothio-cyanatophenyl)amino]thiocarbonyl]-amino]ethyl]amino] carbonyl]methyl]anilino]carbonyl]methyl]phenyl]adenosine), has now been synthesized (14) and was recently characterized in guinea pig heart (15). In the present study we tested its potency in a rabbit heart model of myocardial infarction. To evaluate the potency of m-DITC-ADAC we compared the protection conferred by m-DITC-ADAC with that from both ischemic preconditioning and transient exposure to the A₁AR-selective agonist 2-chloro-N⁶-cyclopentyladenosine (CCPA).

ANIMALS AND METHODS

New Zealand white rabbits of either sex, weighing 1.86 to 2.46 kg, were anesthetized with intravenous sodium pentobarbital (30 mg/kg). All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals, prepared by the United States National Academy of Sciences and published by the National Institutes of Health (NIH publication No. 85-23, revised 1985). The protocols were reviewed and approved by the authors’ institute’s committee for the ethical use of animals. The neck was opened, and a tracheotomy was performed. The rabbits were ventilated with 100% oxygen via a positive pressure respirator (MD Industries, Alabama). Ventilation rate was 30 to 35 breaths/min, and tidal volume was approximately 15 mL. A left thoracotomy was performed in the fourth intercostal space and the pericardium was opened to expose the heart. A 2-0 silk suture on an RB taper needle was passed around a branch of the left coronary artery, and the ends of the suture were threaded through a small vinyl tube to form a snare. The hearts were quickly excised and mounted on a Langendorff apparatus and perfused under 75 mmHg pressure with Krebs buffer containing (in mM) 118.5 sodium chloride, 24.3 sodium bicarbonate, 4.7 potassium chloride, 1.2 magnesium sulphate, 1.2 potassium dihydrogen phosphate, 2.5 calcium chloride and 10 glucose. The Krebs buffer was gassed with 95% oxygen plus 5% carbon dioxide, which results in a pH of 7.4 to 7.5 and PO₂ of 500 to 600 mmHg. The temperature of the perfusate was maintained at 37°C, and the buffer was not recirculated. A fluid-filled latex balloon connected to a pressure transducer via PE240 tubing was inserted into the left ventricle. Balloon volume was adjusted for a systolic pressure of near 100 mmHg. Left ventricular diastolic pressure was not allowed to exceed 15 mmHg. Total coronary artery flow was measured by timed collection of perfusate exiting the heart with a graduated cylinder. Regional ischemia was effected by pulling the snare tight and clamping the tube with a hemostat. Global ischemia was performed by clamping the inflow tubing of the Langendorff apparatus. Reperfusion was achieved by releasing the snare. All hearts were allowed to equilibrate for 20 mins before the experiments were started. Heart rate, left ventricular developed pressure (LVDP) and coronary artery flow were recorded just before and at the end of each drug infusion. One minute before the onset of the 30 min period of ischemia, the hearts were paced at 200 beats/min (4 V/4 ms pulses) until the end of the study.

Measurement of infarct and risk area

At the end of each experiment, the silk suture under the coronary branch was tightly tied to reocclude the artery, and a 0.5% suspension of fluorescent particles (1 to 10 μm diameter, Duke Scientific Corp, California) was infused into the perfusate to mark the risk zone as the nonfluorescent tissue. The hearts were then removed from the Langendorff apparatus, weighed and frozen. While still frozen the hearts were cut into 2 mm transverse slices and the slices were incubated in 1% triphenyl tetrazolium chloride (TTC) in pH 7.4 buffer for 20 mins at 37°C. NAD-dependent dehydrogenases convert TTC to a formazan pigment and stain tissue that still have the enzymes and cofactor a deep red colour. The infarcted tissue loses those constituents and thus does not stain. After staining, the area of infarcted tissue (TTC-negative tissue) and the risk zone (area lacking fluorescence under ultraviolet light) in each slice was traced onto sheets of clear acetate. The areas of both infarct and risk zone in each slice were determined by planimetry. The volumes of infarcted myocardium and myocardium at risk were then calculated by summing the planimetered areas of each and multiplying by the slice thickness. Because the extent of infarction is underestimated when risk zone size is small in rabbit hearts (16,17), data were excluded if the risk zone was found to be less than 0.4 cm^3.

Chemicals

CCPA and DPCPX (8-cyclopentyl-l,3-dipropylxanthine) were purchased from Research Biochemicals International (Massachusetts). m-DITC-ADAC was synthesized as described (14). Compounds were dissolved in DMSO at a concentration of 5 mM, except for m-DITC-ADAC, which was 1 mM. The stock solutions were diluted in Krebs buffer to their final concentration. Stock solutions were stored at 0°C for up to three days except for m-DITC-ADAC, which was stored at −20°C for no more than two weeks.

Experiment protocols

The animals were divided into five groups. The hearts in all groups were subjected to a 30 min coronary branch occlusion followed by 120 mins of reperfusion. The control group (n=8) experienced only the 30 mins of regional ischemia, while the preconditioning group (n= 10) received 5 mins of global ischemia plus 10 mins of reperfusion before the 30 min occlusion. In the CCPA group (n=6) the hearts were exposed to 200 nM CCPA for 5 mins followed by a 10 min washout as a substitute for ischemic preconditioning. In the DPCPX and DPCPX/CCPA group (n=6), 200 nM DPCPX was included in the perfusate for 15 mins starting 20 mins before ischemia. Thus, in the DPCPX/CCPA group the DPCPX was present in the perfusate starting 5 mins before and continued 5 mins after exposure to CCPA. The hearts in the m-DITC-ADAC group (n=5) were perfused with buffer containing 1 μΜ m-DITC-ADAC for 15 mins followed by 10 mins of drug-free perfusion before the 30 min ischemic period was started. Data were obtained from 35 rabbits. All hearts used in the study were perfused with buffer within 1 min after excision.

Statistics

All results are expressed as group mean ± SEM. The differences among groups were determined by a one-way ANOVA with a Newman-Keuls post-hoc test. P<0.05 was considered to be significant. A paired t test was used to test for a drug effect on hemodynamic parameters.

RESULTS

Hemodynamic data

Hemodynamic data for all the groups are summarized in Table 1. Heart rate, LVDP and coronary artery flow from all groups were equivalent before treatment except for a significantly lower heart rate in the CCPA group and a higher coronary flow in the DPCPX/CCPA group. Heart rate was significantly decreased (P<0.01) by 200 nM CCPA, a highly selective A₁AR agonist, and this effect was completely reversed after 10 mins of drug-free perfusion. The effect of CCPA on heart rate was completely abolished when it was accompanied with 200 nM DPCPX, a highly selective A₁AR antagonist that is not thought to affect A₃AR at this concentration (18).

TABLE 1.

Hemodynamic data from isolated rabbit hearts

	Pre-ischemia			Treatment			Washout			30 min reperfusion
Group	HR	CAP	DP	HR	CAF	DP	HR	CAF	DP	HR	CAF	DP
Control	206±7	7.0±0.3	108±5	–	–	–	–	–	–	208±5	5.4±0.3	92±6
PC	203±3	7.4±0.3	99±4	–	–	–	205±3	4.7±0.5	64±6	203±2	5.5±0.3	74±4
CCPA	176±1	7.4±0.3	113±8	123±4*	7.1±0.7	102±9	189±7	7.3±0.5	107±7	200±0	3.9±0.9	59±8
DP-CC	208±7	9.3±0.5	121±7	201±7	7.4±0.6	115±7	196±8	7.4±0.6	121±7	200±4	5.0±0.4	60±6
m-DITC-ADAC	189±8	7.9±0.4	93±7	161±7	7.9±0.4	85±13	143±7*	7.4±0.2	99±9	200±0	7.1±0.8	64±8

CAF Coronary artery flow (mL/min/g); CCPA 2-chloro-N⁶-cyclopentyladenosine; DP Developed pressure (mmHg); DP-CC DPCPX (8-cyclopentyl-1,3-dipropylxanthine) + CCPA; HR Heart rate (beats/min); PC Preconditioned.

^*P<0.05 versus pre-ischemia value

Exposure to the irreversible A₁AR agonist m-DITC-ADAC (1 μΜ for 15 mins) caused a progressive slowing of the heart rate, which did not reach significance (P=0.057) at the end of the 5 min infusion but was significant 10 mins later (P<0.02). Heart rate remained depressed in these hearts throughout the experiment indicating that the effect was indeed irreversible. None of the treatments significantly altered LVDP or coronary flow at the doses used.

Infarct size

Infarct size data are shown in Table 2. There were no significant differences in the area at risk between the groups. Infarct size was normalized by expressing it as a percentage of the area at risk (the field of the occluded artery). Thirty minutes of coronary branch occusion caused 32±2% of the risk zone to be infarcted in control hearts while only 9±2% infarction was seen in the hearts that were preconditioned with 5 mins global ischemia followed by 10 mins reperfusion (P<0.05 versus control). Exposure to 200 nM CCPA for 5 mins protected the hearts as well as ischemic preconditioning with only 13±7% infarction of the risk zone (P<0.05 versus control). As expected, protection conferred by CCPA was completely blocked by 200 nM DPCPX (34±7% infarction, P<0.05 versus CCPA). m-DITC-ADAC, which irreversibly stimulates the A₁AR, also protected the hearts with only 15±4% infarction (P<0.05 versus control) (Figure 1). When pacing was discontinued at the end of the study, hearts treated with m-DITC-ADAC were profoundly bradycardic with a mean rate of 123±9 beats/min. Whether DPCPX or DMSO alone can alter infarct size was not tested in these experiments; both of those tests were conducted in an earlier study from the authors’ laboratory, and no effect was found (8).

TABLE 2.

Infarct size data from isolated rabbit hearts

Group	infarct size (cm3)	Risk size (cm3)	Infarction (%)
Control (n= 8)	0.26±0.02	0.81±0.06	32±2
PC (n=10)	0.07±0.02	0.81±0.08	9±2*
CCPA (n=6)	0.10±0.05	0.69±0.08	13±7*
DP-CC (n=6)	0.25±0.06	0.69±0.06	34±7†
DITC-ADAC (n=5)	0.13±0.03	0.87±0.04	15±4*

^*P<0.05 versus control;

^†P<0.05 versus CCPA (2-chloro-N⁶-cyclopentyladen- osine). DP-CC DPCPX (8-cyclopentyl-1,3-dipropylxanthine) ⁺ CCPA; PC Preconditioned

Figure 1.

Plot showing percentage infarction of the risk region for all of the hearts in the study. Open circles depict individual hearts; closed circles represent group means ± SEM. CCPA 2-chloro-N⁶ -cyclopentyladenosine; CONT Control;· DPCPX 8-cyclopentyl-1,3-dipropylxanthine; PC Preconditioned.

DISCUSSION

The results indicate that CCPA, a highly selective agonist of the A₁AR, could completely mimic protection conferred by ischemic preconditioning in the isolated rabbit heart. Because the A₁AR-selective antagonist DPCPX could block that protection, it was confirmed that the A_lAR was responsible for causing the protection. The irreversible A_lAR-selective agonist m-DITC-ADAC was equally protective. We conclude that a single intracoronary treatment with m-DITC-ADAC should be capable of providing an extended window of protection to the heart against infarction.

m-DITC-ADAC is thought to be an irreversible A_lAR- selective agonist. Treatment of smooth muscle (DDT₁ MF-2) cells in culture or guinea pig isolated hearts with m-DITC-ADAC has been found to elicit a selective, prolonged and irreversible A_lAR agonist effect (15). This activation is not reversed by xanthines due to a presumed covalent attachment of the ligand to the receptor. When m-DITC-ADAC was given to produce a level of bradycardia comparable with that achieved with CCPA, a similar degree of protection was obtained. Because by the activation of A_lAR m-DITC-ADAC is irreversible, it was not appropriate to test whether DPCPX could block the protection. These results support our hypothesis that A₁ARs are present on the heart and that their activation will mimic the preconditioning response.

One of the peculiar properties of ischemic preconditioning is its memory. As was illustrated with CCPA in the present study, the adenosine receptors need only be activated transiently in order to cause the heart to enter a preconditioned state which persists after the agonist has been withdrawn (2). The exact nature of this memory is not known but is thought to be related to an up-regulation of protein kinase C, since inhibitors of this kinase completely reverse the preconditioned state but have no effect on infarction in nonpreconditioned hearts (19). Unfortunately, this memory lasts for only about half an hour (10,11,13) making it difficult to utilize the protection from preconditioning clinically.

Chronic intravenous infusion of CCPA extended the window of protection to at least 6 h (20), but was accompanied by unacceptable side effects including bradycardia and hypotension. Furthermore, by 72 h the protection from a continuous infusion of CCPA was lost due to tolerance (20), and follow-up studies revealed that the site of this tolerance was the adenosine receptor itself (unpublished data). Because of limitations with the isolated heart model we were unable to test how long the protection from a single dose of m-DITC-ADAC will last, but in the present study heart rate remained bradycardic for at least 3 h following a single dose of m-DITC-ADAC and showed no sign of recovery. It is not known how rapidly A_lARs turn over in the heart, but it is possible that a single intracoronary infusion of m-DITC-ADAC could provide several days of protection with few peripheral side effects. In certain high risk patients such treatment could be of value. On the other hand the sensation of angina has been proposed to be mediated by adenosine receptors in the heart (21), and it is possible that intracoronary m-DITC-ADAC could result in intractable anginalike pain.

We (8) and others (9) have recently proposed that the A₃AR also may participate in ischemic preconditioning. That theory was based on the observation that 200 nM DPCPX could not block protection from ischemic preconditioning or from adenosine while the nonselective adenosine antagonist 8-p-sulphophenyl theophylline blocked the protection. An important property of DPCPX is that it is a poor ligand of A₃AR with a reported K_i of 50 μΜ (22). The highly Α₁-selective agonist CCPA also has very poor affinity for the A₃AR (23). The K_i values for inhibition of radioligand binding at rat adenosine receptors for CCPA are 0.6 nM for A₁AR and 237 nM for A₃AR. Protection from 200 nM CCPA was completely blocked by 200 nM DPCPX. The fact that bradycardia, an A₁AR effect (24), was also blocked further confirms that the A_lARs were completely blocked. In light of the above it seems likely that CCPA was able to protect these hearts exclusively through the A₁AR.

If A₃AR were activated by CCPA, then DPCPX would not be expected to have blocked the resulting protection. Indeed this concentration of DPCPX will not block protection from either 10 μΜ adenosine, 65 nM N⁶-[2-(4-amino-phenyl)ethyl]adenosine, a potent mixed A₁ and A₃ agonist or ischemic preconditioning itself. If this conclusion is correct then it would provide evidence that A₁AR and A₃AR, both of which couple to G_i, are present on the rabbit heart and constitute a parallel pathway for preconditioning. m-DITC-ADAC is also not thought to bind to A₃AR, since its precursor, the amine congener ADAC, has roughly the same affinity as CCPA at rat A₁ and A₃ receptors (23).

CONCLUSION

m-DITC-ADAC, a potent irreversible agonist of the adenosine A₁AR, was able to mimic fully the protective effects of ischemic preconditioning in the rabbit heart. An irreversible A₁AR agonist may be useful for extending the duration of protection and minimizing side effects from pharmacological preconditioning.