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Am J Physiol Heart Circ Physiol 284: H859-H866, 2003; doi:10.1152/ajpheart.00251.2002
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Vol. 284, Issue 3, H859-H866, March 2003

A1 adenosine receptor overexpression attenuates ischemia-reperfusion-induced apoptosis and caspase 3 activity

Sara E. Regan1,4, Michael Broad2, Anne M. Byford1,4, Amy R. Lankford1,4, Rachael J. Cerniway1, Marty W. Mayo3, and G. Paul Matherne1,4

Departments of 1 Pediatrics, 2 Surgery, and 3 Biochemistry and Molecular Genetics, and the 4 Cardiovascular Research Center, University of Virginia Health System, Charlottesville, Virginia 22908


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We tested the hypothesis that myocardial ischemia-reperfusion (I/R)-induced apoptosis is attenuated in transgenic mice overexpressing cardiac A1 adenosine receptors. Isolated hearts from transgenic (TG, n = 19) and wild-type (WT, n = 22) mice underwent 30 min of ischemia and 2 h of reperfusion, with evaluation of apoptosis, caspase 3 activity, function, and necrosis. I/R-induced apoptosis was attenuated in TG hearts. TG hearts had less I/R-induced apoptotic nuclei (0.88 ± 0.10% vs. 4.22 ± 0.24% terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells in WT, P < 0.05), less DNA fragmentation (3.30 ± 0.38-fold vs. 4.90 ± 0.39-fold over control in WT, P < 0.05), and less I/R-induced caspase 3 activity (145 ± 25% over nonischemic control vs. 234 ± 31% in WT, P < 0.05). TG hearts also had improved recovery of function and less necrosis than WT hearts. In TG hearts pretreated with LY-294002 (3 µM) to evaluate the role of phosphosinositol-3-kinase in acute signaling, there was no change in the functional protection or apoptotic response to I/R. These data suggest that cardioprotection with transgenic overexpression of A1 adenosine receptors involves attenuation of I/R-induced apoptosis that does not involve acute signaling through phosphoinositol-3-kinase.

cardioprotection; ischemia-reperfusion injury; transgenic mice; phosphoinositol-3-kinase


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

APOPTOSIS appears to be an important mechanism of cell death after ischemia-reperfusion (I/R) injury. In contrast with necrosis, apoptosis is a "programmed cell death" signaling pathway triggered by mitochondrial stress, which results in cytochrome c release and activation of caspases that cause controlled cell destruction. Apoptosis is important in the cellular response to I/R injury, because even small reductions in apoptosis may result in significant myocardial salvage over time (23, 24). Intrinsic cardioprotective mechanisms like ischemic preconditioning, activation of the mitochondrial ATP-sensitive potassium (KATP) channel (2), activation of mitogen-activated protein kinases (MAPK) (1), and activation of phosphoinositol-3-kinase (PI3K) (25) provide increased tolerance to I/R injury and reduce necrotic and apoptotic cell death. We have been studying a model of intrinsic cardioprotection with transgenic overexpression of A1 adenosine receptors in mice hearts. These hearts demonstrate improved tolerance to ischemia as indicated by decreased cell necrosis, improved myocardial energetics, and improved recovery of function (16).

The mechanism of protection from ischemia with A1 receptor overexpression is secondary to A1 signaling through endogenously released adenosine at the time of I/R (18) and involves mitochondrial KATP channels (9) and MAPKs (11, 13). A1 adenosine receptors may also signal via PI3K (25), another important signaling intermediate that may provide the link to the MAPK pathways through protein kinase C (10). Other cardioprotection pathways that involve mitochondrial KATP channels (2) and activation of MAPK (14) have been shown to attenuate I/R-induced apoptosis. Because A1 receptor overexpression also involves these pathways, it is likely that this model inhibits apoptosis associated with I/R injury.

Reductions in I/R-induced apoptosis with A1 adenosine receptor overexpression could be related to acute signaling associated with endogenous adenosine release and A1 receptor activation, similar to the protection from necrosis. Protection from apoptosis could also be the result of a cardioprotective phenotype, conferred by overexpression of A1 receptors, which is similar to adenosine-mediated delayed preconditioning (4). Both MAPK and PI3K have been implicated in inducible cardioprotection pathways (6). In particular, PI3K activation results in activation of Akt, a cell survival signal that attenuates apoptosis through activation of nuclear factor-kappa B, a transcription factor that regulates antiapoptotic proteins (15, 27).

We investigated I/R-induced apoptosis in mouse hearts with A1 adenosine receptor overexpression and showed that cardiac A1 receptor overexpression attenuates I/R-induced apoptosis. Because caspase 3 activation is pivotal for executing the later stages of apoptosis, we also evaluated I/R-induced changes in caspase 3 activity and found that transgenic (TG) hearts with A1 receptor overexpression had less I/R-induced caspase 3 activity. Finally, we examined the role of PI3K in the acute signaling with transgenic A1 adenosine receptor overexpression. With the use of the PI3K inhibitor LY-294002, we showed that inhibition of PI3K did not block the functional recovery or the apoptotic response to I/R injury in TG or WT hearts. In summary, these data suggest that cardioprotection with TG overexpression of A1 adenosine receptors involves attenuation of I/R-induced apoptosis and caspase 3 activity, but does not involve acute signaling through the PI3K pathway. Our findings support the hypothesis that a cardiac protective phenotype is invoked with A1 adenosine receptor overexpression, resulting in reduced I/R-induced apoptosis and caspase 3 activity.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

TG Mouse Model

All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, Revised 1996) and approved through the Institutional Animal Care and Use Committee. TG mice were constructed by using rat A1 adenosine receptor cDNA under the control of the alpha -myosin heavy chain promoter as detailed previously (16). The TG lines used in this study have a maximum A1 adenosine receptor binding capacity of ~2,600-3,300 fmol/mg protein, which corresponds to a 300- to 400-fold receptor overexpression (20).

Isolated Heart Experiments

Langendorff heart model. The cellular and functional responses to I/R were assessed in mice by using an isolated perfused heart model as previously described (5). After anesthesia was administered, hearts from equal numbers of male and female adult mice (10-24 wk old) were excised via thoracotomy and placed into heparinized ice-cold perfusion buffer. After rapid aortic cannulation with a 20-gauge blunt needle, retrograde coronary perfusion was initiated at constant pressure of 80 mmHg using modified Krebs bicarbonate buffer containing (in mM) 118 NaCl, 25 NaHCO3, 4.7 KCl, 1.2 KH2PO4, 2.5 CaCl2, 1.2 Mg2SO4, 11 glucose, and 0.5 EDTA, and equilibrated with 95% O2-5% CO2 at 37°C, giving a pH of 7.4 and a PO2 of ~550 mmHg. The left ventricle was vented with a small polyethylene apical drain. A fluid-filled balloon made of plastic film was inserted into the left ventricle via the mitral valve and inflated to yield a left ventricular diastolic pressure of 3-7 mmHg. The heart was immersed in a fluid-filled bath maintained at 37°C. Continuous left ventricular pressure was measured by connecting the balloon to a pressure transducer. Coronary flow was continuously monitored via a Doppler flow probe (Transonic Systems; Ithaca, NY) located in the aortic perfusion line.

I/R experimental protocol. Hearts were allowed to stabilize at an intrinsic heart rate for 20 min. Pacing at a rate of ~425 beats/min was then initiated to ensure comparable heart rates in all groups. After 10 min of pacing, baseline functional measurements were recorded. In I/R experiments [TG, n = 19; wild type (WT), n = 22], global ischemia was produced by clamping the aortic cannula while simultaneously bubbling the bathing perfusate with 95% N2-5% CO2 to reduce PO2. Pacing was stopped during ischemia. Afer 30 min of normothermic ischemia, reperfusion was initiated by unclamping the aortic cannula and discontinuing nitrogen bubbling. Ventricular pacing was resumed after 2 min of reperfusion, and hearts were reperfused for 120 min to ensure that the onset of apoptotic cell death was initiated (17). Diastolic and systolic pressures, heart rate, and coronary flow were recorded throughout the experimental protocol, and coronary effluent was continuously collected for lactate dehydrogenase assay, an indicator of cell death. To establish baseline apoptosis in an isolated heart model before the effects of I/R, control hearts (TG, n = 9; WT, n = 9) were subjected to the same preischemia protocol. Hearts from a subset of experiments were evaluated for apoptosis by using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining, quantitative DNA fragmentation (nucleosome) assay, and caspase 3 activity. In a separate set of I/R experiments, hearts (WT, n = 9; TG, n = 9) were treated with 3 µM LY-294002, a PI3K inhibitor, for 20 min immediately preceding global ischemia (25). Functional data were recorded throughout, coronary effluent was continuously collected for lactate dehydrogenase assay for cell necrosis, and hearts were evaluated for apoptosis by using quantitative DNA fragmentation assay.

Detection of Apoptosis

Apoptotic myocardial nuclei were identified histologically using TUNEL. Immediately after the experiments, hearts were fixed, dehydrated (with 70% ethanol, 95% ethanol, 100% ethanol, and then xylene), embedded in paraffin, and sectioned at 5 µm thickness. Sections were prepared using the ApopTag TUNEL kit (Intergen; Purchase, NY). Slides were examined under an Olympus BX41 light microscope. With the use of left ventricular regions where myocytes are cut in cross section, 10 fields were examined at ×400, and the number of TUNEL-positive myocyte nuclei was reported as a percentage of total myocyte nuclei counted.

DNA Fragmentation

Myocardial cell apoptosis was also confirmed by quantitating nucleosomal DNA fragmentation in a subset of hearts from the I/R; LY-294002-treated, I/R; and control experiments. Hearts were snap frozen in liquid N2 and stored at -80°C until assayed. Heart samples were dounce homogenized in 2.5 vol of cell lysis buffer (RIPA, in µmol/l: 1 DTT and 50 PMSF) and centrifuged at 13,000 g for 10 min, and supernatant protein concentration was determined by fluorescamine assay. Cell lysate (100 µg) from each heart was used in a photometric enzyme immunoassay for the quantitative determination of cytoplasmic histone-associated-DNA fragments (mono- and oligonucleosomes) of programmed cell death using the commercial assay kit Cell Death Detection ELISAPLUS (Roche; Indianapolis, IN). Results were normalized to the standard provided in the kit and expressed as a fold increase over control.

Capase 3 Activity

Caspase 3 is derived from a proenzyme at the onset of apoptosis and plays an important role in the final common pathway of programmed cell death. During I/R injury, increased caspase 3 activity is indicative of increased programmed cell death signal. Caspase 3 activity was measured in a subset of hearts from I/R and control experiments. To detect caspase 3 activity, 150 µg of lysate from each heart was combined with fluorogenic caspase 3 substrate, diluted to 300 mg/l in caspase assay buffer (250 mmol/l PIPES, 50 mmol/l EDTA, 2.5% CHAPS, and 125 mmol/l DTT), and measured immediately on a fluorometer at an excitation wavelength of 400 nm and an emission wavelength of 505 nm. Measurements were repeated every 10 min for 1 h, the slope of fluorescent units per hour was calculated, and values were compared with known standards to determine enzymatic activity.

Cardiac Function

Left ventricular systolic and end-diastolic pressures (EDP) obtained from a pressure transducer connected to the ventricular balloon were continuously recorded on a MacLab data acquisition system (AD Instruments; Castle Hill, Australia) connected to a Power Macintosh G3 computer. The ventricular pressure signal was digitally processed on-line (using MacLab Chart 3.5.6, AD Instruments) to yield left ventricular developed pressure (LVDP) and heart rate. Ventricular systolic function was evaluated by the measurement of LVDP and expressed as a percentage of baseline plotted over time. Systolic function was also evaluated by an index of total functional recovery, which represents the area under the curve of LVDP plotted over time. Diastolic function was assessed by evaluation of EDP during reperfusion and at the end of reperfusion (final EDP). Coronary flow was continuously monitored via a Doppler flow probe located in the aortic perfusion line.

Detection of Necrosis

To assess the degree of myocardial cell necrosis, efflux of lactate dehydrogenase (LDH) in the coronary effluent was measured. At the onset of reperfusion, coronary effluent was collected continuously from the fluid-filled bath surrounding the heart and pooled throughout the first 60 min of reperfusion. The total volume of pooled effluent was measured and recorded, duplicate 1.5-ml aliquots were taken, and these aliquots were stored at -20°C until analysis. LDH concentration in each sample was measured by using an LDH enzymatic assay kit (Sigma). Total LDH efflux per heart was determined by multiplying LDH concentration in the sample by the total volume of coronary effluent (in liters) collected over 60 min for each heart. Values were normalized for wet heart weight and expressed as LDH units per gram similar to previously reported methods (18).

Statistical Analysis

Data were expressed as means ± SE. Functional parameters were analyzed by multivariate ANOVA for repeated measures and Student-Newman-Keuls post hoc test. Comparisons among groups were made using multivariate ANOVA and Student-Newman-Keuls post hoc test or Student t-test. Statistical significance was accepted for P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Baseline Function

Baseline functional data were recorded and analyzed for all I/R; LY-294002-treated, I/R; and control experiments. No significant sex-related differences were present (data not shown). Baseline functional data for I/R WT (n = 19; TG, n = 22), LY-294002-treated (WT, n = 9; TG, n = 9), and control (WT, n = 9; TG, n = 9) hearts after 30 min of normothermic aerobic perfusion are shown in Table 1. There were no significant differences among groups with regard to heart rate, coronary flow, ventricular function (LVDP), EDP, or wet heart weight (P > 0.05).

                              
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  		Table 1.   Baseline function parameters in Langendorff-perfused hearts from wild-type and transgenic mice

Function After I/R in WT and TG Hearts

Consistent with previous studies, TG hearts overexpressing A1 adenosine receptors demonstrated improved recovery of function following I/R injury, as indicated by better recovery of LVDP over time and by greater final recovery of LVDP (53 ± 2% in TG vs. 29 ± 2% in WT, P < 0.05, Fig. 1A). Values for pressure change over time (dP/dt) reflected changes similar to those observed for LVDP and thus are not reported. Total functional recovery, expressed as a percentage of maximal, was greater in TG versus WT hearts (44 ± 3% vs. 13 ± 1%, respectively; P < 0.05). Ischemic contracture, as indicated by rising EDP, occurred to a greater degree in WT hearts, peaking at 72 ± 4 mmHg in WT hearts compared with 61 ± 3 mmHg in TG hearts (P < 0.05, Fig. 1B). TG hearts showed better recovery of diastolic function throughout reperfusion, with the final EDP of 20 ± 2 versus 39 ± 3 mmHg in WT hearts at the end of 120 min of reperfusion (P < 0.05, Fig. 1B). Coronary flow was similar in TG and WT hearts at preischemia (P > 0.05, Table 1) and throughout reperfusion (P > 0.05, Fig. 1C).


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  		Fig. 1.   A: time course of left ventricular developed pressure in wild-type (WT, n = 22) and transgenic (TG, n = 19) isolated hearts subjected to 30 min of ischemia and 2 h of reperfusion. Values shown are means ± SE. * P < 0.05, TG vs. WT. B: time course of end-diastolic pressure in WT (n = 22) and TG (n = 19) isolated hearts subjected to 30 min of ischemia and 2 h of reperfusion. Values shown are means ± SE. * P < 0.05, WT vs. TG. C: coronary flow in WT (n = 22) and TG (n = 19) isolated hearts subjected to 30 min of ischemia and 2 h of reperfusion. Values shown are means ± SE. P > 0.05, TG vs. WT hearts.

I/R-Induced Necrosis

Tissue viability was better preserved in TG hearts compared with WT hearts. In a subset of I/R experiments, cell death was estimated by total LDH efflux during ischemia and the first 60 min of reperfusion. In WT hearts (n = 7), total LDH efflux was 18.0 ± 2.4 U/g, whereas TG hearts (n = 4) had significantly reduced LDH efflux, 9.4 ± 3.1 U/g (P < 0.05, Fig. 2).


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  		Fig. 2.   Total lactate dehydrogenase (LDH) efflux (U/g wet heart wt) in coronary effluent collected during ischemia and the first hour of reperfusion from WT (n = 4) and TG (n = 4) isolated hearts subjected to 30 min of stabilization followed by 30 min of ischemia and 2 h of reperfusion. Values shown are means ± SE. * P < 0.05, WT vs. TG.

I/R-Induced Apoptosis

TG hearts exhibited less I/R-induced apoptosis compared with WT (Fig. 3). Very few TUNEL-positive myocyte nuclei were observed in control hearts from either group: 0.42 ± 0.12% in WT hearts (n = 4) versus 0.55 ± 0.14% in TG hearts (n = 4), (P = 0.54, Fig. 3). Thirty minutes of global ischemia followed by 2 h of reperfusion increased the number of TUNEL-positive cells in the ventricular myocardium. TG hearts, however, showed substantially less I/R-induced apoptosis compared with WT hearts. WT I/R hearts (n = 7) had 4.21 ± 0.26% TUNEL-positive myocytes versus only 0.88 ± 0.11% in TG I/R hearts (n = 5, P < 0.05, Fig. 3).


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  		Fig. 3.   Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive myocytes in WT control (n = 4) and TG control (n = 4) were compared with WT (n = 7) and TG (n = 5) isolated hearts subjected to 30 min of ischemia and 2 h of reperfusion (I/R). Values shown are means ± SE. ** P < 0.05 vs. WT control; * P < 0.05 vs. WT I/R hearts.

To confirm the apoptosis observed histologically, heart lysates from a subset of experiments were assayed for DNA fragmentation by using a quantitative nucleosome assay. Consistent with the TUNEL results, DNA fragmentation in WT I/R hearts (n = 14) increased 4.90 ± 0.4-fold over control hearts versus only 3.29 ± 0.4-fold increase over control in TG I/R hearts (n = 11, P < 0.05, Fig. 4A). Both methods of detecting apoptosis (TUNEL and DNA fragmentation assay) demonstrate significant attenuation of apoptosis in TG hearts.


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  		Fig. 4.   A: DNA fragmentation at the end of reperfusion in WT (n = 14) and TG (n = 11) isolated hearts subjected to 30 min of ischemia and 2 h of reperfusion. * P < 0.05, TG vs. WT. B: caspase 3 activity for WT (n = 14) and TG (n = 11) isolated hearts subjected to 30 min of ischemia and 2 h of reperfusion. Values shown are means ± SE. * P < 0.05, TG vs. WT.

I/R-Induced Caspase 3 Activity

I/R-induced caspase 3 activity was attenuated in TG hearts compared with WT hearts. Caspase 3 activity in WT hearts (n = 14) increased 234 ± 31% over control hearts versus only 145 ± 25% increase in TG hearts (n = 11, P < 0.05, Fig. 4B).

I/R Injury in Hearts Treated With LY-294002, a PI3K Inhibitor

Inhibition of PI3K with LY-294002 before and during ischemia did not block the improved functional recovery, the decreased stunning, or the apoptotic response to I/R injury in TG hearts. Similar to untreated I/R hearts, TG hearts treated with LY-294002 demonstrated improved recovery of function versus LY-294002-treated WT hearts following I/R, as indicated by final recovery of LVDP (51 ± 3% vs. 29 ± 5% in WT hearts, P < 0.05). Total functional recovery was also greater in LY-294002-treated TG hearts (40 ± 4% vs. 15 ± 4% in LY-294002-treated WT hearts, P < 0.05, Fig. 5A). LY-294002-treated TG hearts also showed better final recovery of diastolic function at the end of 120 min of reperfusion, with an EDP of 18 ± 3 versus 33 ± 3 mmHg in WT LY-294002-treated hearts (P < 0.05, Fig. 5B). Coronary flow was similar in TG and WT hearts treated with LY-294002 before ischemia and throughout reperfusion (P > 0.05, data not shown). Similar to untreated I/R hearts, tissue viability after I/R was better preserved in LY-294002-treated TG hearts compared with LY-294002-treated WT hearts. In LY-294002-treated hearts, WT total LDH efflux was 18.1 ± 2.9 versus 11.0 ± 1.9 U/g in LY-294002-treated TG hearts (P < 0.05, Fig. 5C).


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  		Fig. 5.   A: total functional recovery of left ventricular developed pressure during reperfusion in LY-294002 (LY)-treated (WT, n = 9; TG, n = 9) isolated hearts subjected to 30 min of ischemia and 2 h of reperfusion. Values shown are means ± SE. * P < 0.05, TG vs. WT. B: final left ventricular end-diastolic pressure (expressed in mmHg) at the end of reperfusion in LY-treated (WT, n = 9; TG, n = 9) isolated hearts subjected to 30 min of ischemia and 2 h of reperfusion. Values shown are means ± SE. * P < 0.05, WT vs. TG. C: total LDH efflux (U/g wet heart wt) in coronary effluent collected during the first hour of reperfusion from LY-treated (WT, n = 9; TG, n = 9) isolated hearts subjected to 30 of stabilization followed by 30 min of ischemia and 2 h of reperfusion. Values shown are means ± SE. * P < 0.05, WT vs. TG.

Finally, LY-294002 did not block the protection from I/R-induced apoptosis in TG hearts overexpressing A1 adenosine receptors. After I/R, DNA fragmentation (by nucleosome assay) in LY-294002-treated WT hearts (n = 8) increased 4.61 ± 0.4-fold over hearts versus only a 2.94 ± 0.4-fold increase over control in LY-294002-treated TG I/R hearts (n = 9, P < 0.05, Fig. 6).


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  		Fig. 6.   DNA fragmentation at the end of reperfusion in LY-treated (WT, n = 8; TG, n = 9) isolated hearts subjected to 30 min of ischemia and 2 h of reperfusion. * P < 0.05, TG vs. WT.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we have shown that acute myocardial I/R injury induces apoptosis and that this apoptotic response is attenuated with transgenic A1 adenosine receptor overexpression in the mouse heart. These findings are associated with attenuation of I/R-induced increases in caspase 3 activity and improved acute functional recovery (stunning) in our model. Additionally, inhibition of the PI3K cell signaling pathway with LY-294002 before and during ischemia does not block the A1 adenosine receptor overexpression-mediated reductions in apoptosis, necrosis, or stunning observed in TG mice following I/R injury. These findings suggest that the PI3K pathway may not be involved in the acute signaling process of cardioprotection from I/R injury observed in TG mice with overexpression of A1 adenosine receptors.

In response to ischemia and reperfusion, a myocyte can become dysfunctional (stunned), infarcted (necrotic), or apoptotic. Apoptosis is a fundamental process in cell survival and cell death that occurs via activation of distinct signaling pathways involving mitochondria, mitochondrial regulatory proteins, and activation of caspases. Ultimately, cells undergo nuclear chromatin condensation, DNA fragmentation, and formation of apoptotic bodies (26). Ischemia and reperfusion, but not ischemia alone, has recently been shown to initiate this apoptotic cascade in myocardial cells (8). In isolated rat hearts, 30-min global ischemia followed by 120-min reperfusion resulted in apoptosis in cardiomyocytes, as determined by TUNEL staining (8). Inhibition of the apoptotic response to I/R may be very important in the response to myocardial I/R. Administration of a caspase 3 inhibitor (Ac-DEVD-CHO) during reperfusion significantly improves contractile recovery following an acute ischemic insult in perfused rat hearts (22), and others have shown a reduction in I/R-induced apoptosis with ischemic preconditioning (17).

In the current study, we demonstrate that global I/R in WT mouse hearts results in 4% apoptotic nuclei. Although this seems like a modest amount, a recent investigation suggests that apoptosis following an acute ischemic insult is progressive (3), and therefore small distributions of apoptosis initially may result in significant myocardial cell loss over time (6, 23, 24). Ratcliffe (21) has suggested that the expansion of postischemic noninfarcted dysfuntional myocardium over time may be attributed to a progression of apoptosis. Thus the apoptotic myocytes may represent the most significant injury to the myocardium, because they may be responsible for the dysfunction and myocyte loss observed over many months following myocardial ischemia (19).

In our model of A1 adenosine receptor overexpression, we have demonstrated cardioprotection from I/R-induced stunning (16), necrosis (18), and now apoptosis. The exact mechanisms of this protection are not fully understood. We have shown that the protection from stunning and necrosis involves direct A1 receptor activation (16), MAPK activation (11), and activation of mitochondrial KATP channels (9) and mimics ischemic preconditioning (18). In the current study, we hypothesized that activation of PI3K, which has been shown to mediate the attenuation of apoptosis with ischemic preconditioning (25), may also be involved in the mechanism of cardioprotection with A1 overexpression. However, acute inhibition of PI3K did not block the protection from I/R-induced stunning, necrosis, or apoptosis in our model. These findings suggest that transgenic A1 adenosine receptor overexpression may invoke a cardioprotective phenotype that modifies the I/R-induced apoptotic response. In support of this theory, we have recently shown that transgenic A1 receptor overexpression alters expression of genes and proteins involved in cardioprotection (12). Using microarray analysis, we demonstrated increased gene expression of anti-apoptotic proteins such as BCl-XL and inhibitor of apoptosis proteins (IAPs) and downregulated expression of proapoptotic caspase 8. Additionally, we found that expression of the caspase 8 precursor protein as well as the activated caspase 8 peptides were also decreased in hearts with trangenic A1 receptor overexpression (12). The chronic alteration of expression of these genes and proteins may contribute to the phenotype of attenuation of apoptosis observed in this study.

There are some technical limitations regarding the detection of apoptosis and the inhibition of PI3K. LY-294002 treatment before and during ischemia may inhibit PI3K acutely but would not block chronic PI3K signaling through trangenic A1 adenosine receptor overexpression. Long-term pretreatment with LY-294002 would be required to inhibit the effects of chronic PI3K signaling on cell survival in response to I/R injury in our model.

Regarding the detection of apoptosis, we measured DNA fragmentation using two different techniques that showed different magnitudes of apoptosis. First, apoptotic cells were histologically identified by TUNEL staining of I/R hearts. In the TUNEL assay, TdT binds to the exposed 3'-OH fragmented DNA ends and catalyzes the addition of conjugated deoxynucleotides for identification of apoptotic nuclei. The TUNEL method indicated a nearly fourfold decrease in DNA fragmentation in transgenic hearts. Additionally, ELISA detection of histone-associated DNA fragments (nucleosomes) from lysates of I/R hearts showed a twofold decrease in apoptosis in TG hearts. It has been suggested that the TUNEL method is overly sensitive (7), and for this reason we verified the data by using the ELISA assay. Regardless, both techniques demonstrated that DNA fragmentation in response to I/R was reduced in hearts with transgenic overexpression of A1 adenosine receptors.

In summary, A1 adenosine receptor overexpression attenuates the apoptosis associated with I/R injury and decreases I/R-induced caspase 3 activity. The decreased caspase 3 activity following I/R injury may represent a cardioprotective phenotype invoked by A1 adenosine receptor overexpression. The PI3K pathway does not appear to be involved in the acute signaling mechanism of this protection. Inhibition of the apoptotic response to I/R injury may affect long-term postischemic outcome, and therefore A1 adenosine receptor overexpression may provide significant benefits in the long-term recovery following myocardial infarction.


    ACKNOWLEDGEMENTS

This study was supported by a grant-in-aid from the Children's Medical Center at University of Virginia (6-41770) and by National Heart, Lung, and Blood Institute (NHLBI) Grant RO1HL-59419 (to G. P. Matherne). S. E. Regan was supported by NHLBI Grant T32HL-07956, and G. P. Matherne was supported by NHLBI Independent Scientist Award KO2HL-67823.


    FOOTNOTES

Address for reprint requests and other correspondence: G. P. Matherne, Dept. of Pediatrics, Univ. of Virginia Health System, MR-4 Bldg., Box 801356-1356, Charlottesville, VA 22908 (E-mail: gpm2y{at}virginia.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpheart.00251.2002

Received 11 October 2002; accepted in final form 14 November 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abe, J, Baines CP, and Berk BC. Role of mitogen-activated protein kinases in ischemia and reperfusion injury: the good and the bad. Circ Res 86: 607-609, 2000[Free Full Text].

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Am J Physiol Heart Circ Physiol 284(3):H859-H866
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