Gene Dosage-Dependent Effects of Cardiac-Specific Overexpression of the A3 Adenosine Receptor
We used a genetic approach to determine whether increasing the level of A3 adenosine receptors (A3ARs) expressed in the heart confers protection against ischemia without causing cardiac pathology. We generated mice carrying one (A3tg.1) or six (A3tg.6) copies of a transgene consisting of the cardiomyocyte-specific α-myosin heavy chain gene promoter and the A3AR cDNA. A3tg.1 and A3tg.6 mice expressed 12.7±3.15 and 66.3±9.4 fmol/mg of the high-affinity G protein–coupled form of the A3AR in the myocardium, respectively. Extensive morphological, histological, and functional analyses demonstrated that there were no apparent abnormalities in A3tg.1 transgenic mice compared with nontransgenic mice. In contrast, A3tg.6 mice exhibited dilated hearts, expression of markers of hypertrophy, bradycardia, hypotension, and systolic dysfunction. When A3tg mice were subjected to 30 minutes of coronary occlusion and 24 hours of reperfusion, infarct size was reduced ≈30% in A3tg.1 mice and ≈40% in A3tg.6 mice compared with nontransgenic littermates. The reduction in infarct size in the transgenic mice was not related to differences in risk region size, systemic hemodynamics, or body temperature, indicating that the cardioprotection was a result of increased A3AR signaling in the ischemic myocardium. The results demonstrate that low-level expression of A3ARs in the heart provides effective protection against ischemic injury without detectable adverse effects, whereas higher levels of A3AR expression lead to the development of a dilated cardiomyopathy.
Adenosine increases to high levels in the ischemic myocardium where it acts on cell surface receptors to delay cell death as well as to increase the resistance of the heart to subsequent ischemic insults (ischemic “ preconditioning”). Because of the potent and diverse beneficial actions of adenosine, there is great interest in developing therapies that enhance the actions of this endogenous cardioprotective metabolite. To date, two approaches have been tested extensively. The first is to mimic the actions of adenosine by administering subtype-selective agonists. The second is based on increasing interstitial levels of adenosine in the ischemic myocardium either by altering its metabolism or by preventing its uptake into endothelial cells. Although these pharmacological approaches have proven to be effective in experimental models of ischemia/reperfusion injury, their clinical use is complicated by side effects associated with systemic drug administration and loss of efficacy due to receptor desensitization.
An alternative method to enhance the actions of adenosine involves a molecular genetic approach. Matherne and Headrick1 have recently demonstrated that increasing the level of expression of A1ARs in the heart by cardiac-specific transgenesis increases tolerance to ischemic injury. These results suggest that the endogenous cardioprotective actions of adenosine are not only limited by the quantity of adenosine that is produced, but also by the number of functional receptors expressed in the myocardium. Theoretically, targeted expression of adenosine receptors could overcome many of the obstacles associated with other forms of adenosinergic therapy. Furthermore, a greater cardioprotective response may be expected with this approach. One potential problem associated with chronic overexpression of G protein–coupled receptors, however, is that the heart may become diseased. For example, several previous studies have shown that overexpressing G protein–coupled receptors or their signaling components can lead to ventricular hypertrophy or dilated cardiomyopathy.2
Although the A1AR has been implicated as the primary receptor responsible for mediating the cardiac actions of adenosine, recent studies suggest that A3ARs also play an important cardioprotective role.3–6⇓⇓⇓ Similar to the A1AR, the A3AR inhibits adenylyl cyclase and modulates other signaling pathways regulated by Gi/o proteins.7 In the present study, we tested the hypothesis that cardiac-specific expression of A3ARs enhances tolerance to ischemic injury and that this can be achieved at levels of receptor expression sufficiently low as to not result in adverse effects. This hypothesis was tested by generating transgenic mice expressing the A3AR in the heart at different levels using the cardiomyocyte-specific α-myosin heavy chain (α-MyHC) gene promoter.
Materials and Methods
All procedures were performed in accordance with the guidelines established by the Medical College of Wisconsin and the University of Louisville, which conform to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.
Generation of A3AR Transgenic Mice
Transgenic mice cardiac-specifically overexpressing the A3AR were created using standard techniques.8 Briefly, a linear 8.0-kb DNA fragment (Figure 1) containing the α-MyHC gene promoter, the full-length canine A3 adenosine receptor cDNA, and a polyadenylation signal was released by NotI digestion and microinjected into the pronuclei of fertilized mouse oocytes (FVB/N), which were subsequently transferred to the oviducts of pseudopregnant female FVB/N mice (Taconic, Germantown, NY). The presence of the transgene in F0 mice was detected by PCR analysis using one primer specific for the A3AR (5′-GTCTTGAACTCCCGTCCA-3′) and one primer specific for the α-MyHC gene promoter (5′ -AAGCCTAGCCCACACCAGAAATGACAGACA-3′; Figure 1). Once the founders were identified by PCR, the transgene copy number was determined by Southern blot analysis of genomic DNA isolated from tail samples using a 32P-labeled probe (722-bp EcoRI-XbaI fragment) corresponding to the α-MyHC gene promoter. The probe hybridizes to a 2.6-kb EcoRI fragment diagnostic of the endogenous α-MyHC gene and to a 3.9-kb fragment diagnostic of the α -MyHC/A3AR transgene (Figure 1). To determine the copy number in transgenic mice, the intensity of the endogenous α-MyHC allele (2 copies) was compared with that of the transgene using a Storm phosphoimager and ImageQuant software (Molecular Dynamics).
Radioligand Binding Assays and Adenylyl Cyclase Assays
Radioligand binding assays were performed with membranes prepared from mouse hearts using N6-(4-amino-3-[125I]iodobenzyl)adenosine-5′-N-methylcarboxamide (125I-AB-MECA), as described previously in detail.4,9⇓ For adenylyl cyclase assays, ventricular membranes (≈100 μg) were incubated with 5 mmol/L MgCl2, 0.5 mmol/L EDTA, 0.2 mmol/L ATP, 0.1 mmol/L GTP, 20 μmol/L Ro 21 1724, 1 mmol/L ascorbic acid, 25 mmol/L phosphocreatine, and 1 mg/mL creatine phosphokinase in Tris buffer (20 mol/L [pH 7.4]) for 10 minutes at 37°C. Reactions were terminated by the addition of 500 μL of 0.1 mmol/L HCl. cAMP in the acid extracts was quantitated using radioimmunoassay (Amersham). In both the binding assays and the adenylyl cyclase assays, WRC 0571 (300 nmol/L) was included to inhibit A1ARs.10
mRNA Expression Studies
For Northern blot analysis, 10 μg of left ventricular total RNA was electrophoresed through 1% agarose gels containing 10% formaldehyde and then transferred to nylon membranes. The membranes were hybridized with a full-length canine A3AR 32P-labeled probe.9 For RNA dot blot analyses, 3 μg of left ventricular total RNA was blotted onto nylon membranes using a filtration manifold. Membranes were hybridized with 32P labeled oligonucleotide probes corresponding to atrial natriuretic factor (ANF), α-skeletal actin (α-skel actin), α -myosin heavy chain, β-myosin heavy chain, sarcoplasmic reticulum calcium ATPase isoform-2a (SERCA), phospholamban (PLB), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The probes used for hybridization have been described previously.11 Hybridization signals were quantitated using the phosphoimager. The signal intensity of each blot was normalized to the GAPDH signal to account for loading errors.
The hearts were fixed under pressure in situ by inserting a 23-gauge needle into the apex of the heart of anesthetized mice and perfusing at a pressure of 75 mm Hg with a 4% paraformaldehyde solution for 5 minutes. After embedding the hearts in paraffin, step serial sections (5 μm) were taken and mounted on slides. Adjacent sections were stained with hematoxylin and eosin or with Masson’s trichrome stain and examined microscopically.
Hemodynamic Measurements and Echocardiography
Heart rate and blood pressure were determined in the conscious state using a computerized tail-cuff manometer. Echocardiographic assessment (Toshiba T380 Powervision system) was performed on mice lightly anesthetized with tribromoethanol (20 mg/100 g body weight IP) in the parasternal long-axis, short-axis, and apical 4-chamber views using a custom-made gel-filled acoustic standoff and a pediatric 7.5-MHz broadband transducer effectively operating at 10-MHz frequency. The parasternal views were used to measure anteroposterior internal diameter (D), anterior wall thickness (AWTh), and posterior wall thickness (PWTh) at end-diastole (ED) and end-systole (ES). Left ventricular systolic function was assessed by fractional shortening (FS=[(EDD−ESD)/EDD]), and mean velocity of circumferential shortening (Vcf, circ/sec=FS/ejection time) corrected for heart rate (Vcf divided by the square root of the RR interval in seconds).12
In Vivo Mouse Model of Infarction
The model of ischemia and reperfusion has been described previously in detail.13,14⇓ Briefly, an occluder was placed around the proximal portion of the left anterior descending (LAD) coronary artery under general anesthesia with sodium pentobarbital. Heart rate was determined from ECG recordings taken during the procedure using needle electrodes connected to a computerized data acquisition system. Subsequently, the mice were subjected to 30 minutes of coronary occlusion after which the chest was closed and the mice were allowed to recover for 24 hours. On the following day, infarct size was assessed by staining with triphenyltetrazolium chloride and phthalo blue dye. Infarct size was expressed as a percentage of the area at risk.
Of the 45 mice that were created after microinjection and embryo implantation, 6 were found to carry the transgene with copy numbers of 1, 3, 6, 14, 15, and 20. The 3 founder mice with the highest transgene copy number died within 4 weeks of birth due to massive cardiac enlargement with dilated atria, pulmonary congestion, and pleural effusions. Of the 3 remaining founders, the mouse carrying 3 copies of the transgene could not produce offspring, whereas lines were successfully established for the remaining 2 founders (copy numbers of 1 and 6) by breeding with wild-type FVB/N mice. Thus, 2 lines of heterozygous mice carrying 1 (line A3tg.1) and 6 (line A3tg.6) copies of the transgene were characterized in detail. Figure 1 illustrates the results of Southern and Northern analysis of genomic DNA or total heart RNA, respectively, of F1 A3tg.1 and A3tg.6 mice. These results demonstrate that the transgene is transmitted in the 2 lines of mice and that the A3AR is being expressed in the heart at levels that correlate with the transgene copy number.
Radioligand binding analysis with 125I-AB-MECA demonstrated that the A3tg.1 and A3tg.6 lines exhibit increased expression of the A3AR in the heart. As shown in Figure 2, incubating membranes from A3tg.1 and A3tg.6 hearts with ≈0.3 nmol/L 125I-AB-MECA resulted in specific binding, which was 44% and 73% of total binding, respectively.
Careful analysis of equilibrium binding data revealed that 125I-AB-MECA bound to 2 affinity states in the transgenic mouse heart membranes, which is clearly illustrated following Scatchard transformation of the data (Figure 2). Bmax values for both the high- and low-affinity sites were calculated to be 12.7±3.15 and 85.5±9.37 fmol/mg protein for A3tg.1 hearts (total Bmax= 98.2±10.3 fmol/mg protein) and 66.3±9.4 and 192±16.5 fmol/mg protein for A3tg.6 hearts (total Bmax=258±27.1 fmol/mg protein). The Kd values of 125I-AB-MECA for the 2 affinity sites were similar in A3tg.1 and A3tg.6 hearts (Table 1). In previous studies,9 we have demonstrated that the high-affinity site represents binding of 125I-AB-MECA to the G protein–coupled form of the A3AR and the low-affinity site represents binding to the uncoupled form of the receptor, allowing for the determination of the number of receptors that are G protein–coupled. Based on this, we calculated that 12.9% of the A3ARs are coupled to G proteins in A3tg.1 mice whereas 25.7% of the receptors are coupled in A3tg.6 mice. Moreover, the number of A3ARs coupled to G proteins that are expressed in A3tg.6 mice is 5.2-fold higher than in A3tg.1 mice (Table 1).
A3ARs inhibit adenylyl cyclase via pertussis toxin–sensitive inhibitory Gi/o proteins.15 Therefore, we assessed the adenylyl cyclase system in nontransgenic, A3tg.1, and A3tg.6 mouse heart membranes. Basal adenylyl cyclase activities as well as isoproterenol- or forskolin-stimulated adenylyl cyclase activities were similar in the 3 groups of mice (Table 1). During costimulation with IB-MECA (1 μmol/L), isoproterenol- and forskolin-induced adenylyl cyclase activity was significantly reduced in A3tg.1 and A3tg.6 hearts, but not in nontransgenic hearts. These results confirm that A3ARs expressed in A3tg.1 and A3tg.6 mice are functionally coupled to Gi/o proteins.
Pathological and Functional Characterization
Characterization of A3tg.1, A3tg.6, and nontransgenic mice was carried out at 14 to 17 weeks of age. In general, there were no differences between the 3 lines of mice in their appearance or in their body weights (Table 1). However, gross morphological analysis revealed an ≈20% increase in the heart weight of A3tg.6 mice compared with A3tg.1 mice and nontransgenic littermate mice (Table 1). By visual inspection, the right and left atria of A3tg.6 mice were markedly enlarged. Ventricular weights were also moderately increased in A3tg.6 mice (Table 1). The increased heart size in A3tg.6 mice was not the result of increased arterial pressure, because heart rate and blood pressure assessed in the conscious state by the tail-cuff manometer were, in fact, decreased in A3tg.6 mice (Table 1). Microscopic examination of ventricular tissue sections revealed the presence of myocardial disarray in A3tg.6 mice (Figure 3). However, fibrosis was not evident up to 28 weeks of age, as assessed by Masson’s trichrome stain (data not shown). Finally, there was increased mRNA expression of fetal genes in the ventricles of A3tg.6 mice, including ANF, α -skeletal actin, and β-MyHC, as well as decreased expression of α-MyHC and SERCA-2a (Table 1 and Figure 3). When normalized to the levels of GAPDH, ANF and β-MyHC exhibited the greatest increase in expression (by ≈8.8- and 4.7-fold, respectively). Expression of α-MyHC and SERCA-2a were both decreased 50%. Overall, no gross morphological, microscopic, or molecular differences were observed between nontransgenic and A3tg.1 mice (Table 1).
Echocardiographic analysis provided further evidence for the development of pathology in A3tg.6 mice. M-mode measurements included the left ventricular minor axis dimensions at end-diastole (EDD) and end-systole (ESD) and the thickness of the anterior (EDAWTh) and posterior (EDPWTh) walls. Representative M-mode echocardiograms from nontransgenic, A3tg.1, and A3tg.6 mice are shown in Figure 4, and group data (n=8/group) are presented in Table 1. There were no differences in EDD, ESD, EDAWTh, EDPWTh, or fractional shortening between nontransgenic and A3tg.1 mice. In A3tg.6 mice, however, left ventricular dimensions were significantly increased 15 to 30% and fractional shortening was significantly reduced ≈20% (Table 1 and Figure 4). Additionally, absolute wall thickness was mildly increased without changes in relative wall thickness, indicating slight concentric ventricular hypertrophy (Table 1). Because heart rate was decreased in A3tg.6 mice (Table 1), we normalized Vcf to the RR interval. The corrected Vcf was reduced 44% in A3tg.6 mice (Table 1), indicating significant systolic dysfunction. The reduction in Vcf occurred despite mild reductions in systolic blood pressure and afterload.
A3tg.1, A3tg.6, and age-matched (14.8±0.5 weeks) nontransgenic littermate mice were assessed with respect to their susceptibility to infarction using an in vivo model of ischemia/reperfusion injury.13,14⇓ Subjecting nontransgenic littermate mice to 30 minutes of left anterior descending coronary artery occlusion followed by 24 hours of reperfusion resulted in significant infarction, as detected by macroscopic histochemical staining with triphenyltetrazolium chloride (Figure 5). When expressed as a percentage of the risk region, infarct size averaged 48.1±2.3% in nontransgenic mice. In contrast, infarct size after an identical ischemic insult was 35.1±3.6% of the risk region in A3tg.1 transgenic mice and only 28.5±3.0% of the risk region in A3tg.6 mice. Thus, the infarcts were ≈ 30% smaller in A3tg.1 transgenic mice and ≈40% smaller in A3tg.6 transgenic mice compared with nontransgenic littermates (P<0.05 by 1-way ANOVA). The reductions in infarct size in the A3AR transgenic mice were not related to differences between the groups with respect to risk region size (42.6±0.9%, 41.3±1.8%, and 41.0±1.8% in nontransgenic, A3tg.1, and A3tg.6 mice, respectively) or core body temperature (data not shown). Heart rate was significantly lower (≈30%) in A3tg.6 mice during the experiments (Table 2). However, heart rate was not altered in A3tg.1 mice, suggesting that the reductions in infarct size observed in the transgenic mice were also not related to differences between the groups with respect to systemic hemodynamics.
The results of the present study demonstrate that it is possible to increase the number of functional A3ARs in cardiac myocytes of transgenic mice and that this manipulation has a beneficial effect resulting in enhanced resistance to ischemia/reperfusion injury. In A3tg.1 mice, which carry a single copy of the α -MyHC/A3AR transgene expressing 12.7±3.15 fmol/mg of the high-affinity G protein–coupled form of the A3AR in the myocardium, infarct size after regional myocardial ischemia and reperfusion was reduced ≈30% compared with nontransgenic littermates. In mice carrying higher transgene copy numbers, however, severe adverse effects were observed. A3tg.6 mice, which carry 6 copies of the transgene and express 66.3±9.4 fmol/mg of the high-affinity form of the A3AR, developed a dilated cardiomyopathy characterized by increased heart mass, multichamber dilation, expression of markers of hypertrophy, bradycardia, hypotension, and systolic dysfunction. In addition, mice with more than 6 copies of the transgene died prematurely and exhibited overt signs of heart failure. Despite the presence of cardiac abnormalities, however, infarct size after ischemia/reperfusion injury was further reduced in A3tg.6 mice. Taken together, these results indicate that in transgenic mice cardiac-specifically overexpressing A3ARs, two phenotypes develop in a gene dosage-dependent manner, a cardioprotected phenotype that appears at relatively low levels of receptor expression and a cardiomyopathic phenotype that develops when A3ARs are expressed at high levels.
The infarct-limiting effects observed in A3tg mice were not related to differences in systemic hemodynamics, risk region size, or core body temperature, three primary determinants of infarct size. In addition, we have determined in preliminary studies using an isolated heart model that functional recovery after global ischemia and reperfusion is improved in A3tg.1 mouse hearts and that the A3AR antagonist MRS 1220 abrogates this protective effect.16 These findings imply that the cardioprotection observed in A3tg hearts is receptor-mediated and results from increased signaling via the A3AR. Thus, in agreement with previous results obtained with A1AR transgenic mice,1 as well as the results of recent studies by Dougherty et al17 that showed that increased expression of the A3AR in isolated cardiomyocytes protects against cellular injury caused by simulated ischemia and reperfusion, our data indicate that the number of receptors that are normally expressed in the myocardium limits the cardioprotective effect of adenosine produced endogenously during ischemia. Previous studies have demonstrated that inhibitors of protein kinase C and of the ATP-dependent potassium channel block the cardioprotective effects of adenosine receptor agonists as well as those of ischemic preconditioning.18 It seems reasonable, therefore, to propose that increasing the level of expression of A3ARs in cardiomyocytes to moderate levels magnifies these signaling pathways leading to an enhanced cardioprotective effect.
On the other hand, the results obtained with mice carrying higher transgene copy numbers suggest that basal signaling is increased when the A3AR is expressed at higher levels, leading to the development of a multichambered dilated cardiomyopathy. This may be the result of increased abundance of constitutively activated A3ARs, magnification of the effects of low concentrations of adenosine that are normally present in the myocardium, and/or alteration of other signaling pathways due to disruption of the stoichiometry between receptors and G proteins. Although the exact mechanisms leading to the cardiomyopathic phenotype in A3tg.6 mice remain unknown, we predict that it is the result of enhanced Gi signaling. This hypothesis is based on the studies of Redfern and colleagues,19 who recently characterized a severe dilated cardiomyopathic phenotype similar to that observed in the present investigation in transgenic mice overexpressing a modified form of the κ opioid receptor. This hypothesis is further supported by the observation that heart weights are increased in A1AR transgenic mice expressing very high levels of this Gi protein–coupled receptor.20 The possibility remains, however, that the abnormal phenotype observed in A3tg.6 mice is the result of enhanced activation of other signaling pathways implicated in myocardial adaptive responses such as Gq proteins21,22⇓ or the small G protein Rho A.23,24⇓
It should be noted that we only measured the level of expression of the A3AR in the present studies at a single time point (14 to 17 weeks of age). Because of reduced activity of the α-MyHC gene promoter in response to the progression of disease, it is likely that the level of expression of the A3AR in A3tg.6 mice decreased with age. If this is the case, the degree of expression of the A3AR needed to generate cardiac dysfunction may actually be greater than the present binding data indicate. It should also be noted that we were not able to detect the expression of A3ARs in wild-type mice by radioligand binding analysis or by Northern blot analysis, suggesting that A3ARs may not be expressed endogenously in the murine myocardium. However, previous studies in dogs, rabbits, and rats4,9,25⇓⇓ have demonstrated that A3AR transcripts in the heart can only be detected by RT-PCR, suggesting that they are expressed at low levels requiring very sensitive techniques for detection. A3ARs are generally considered to be expressed in cardiac myocytes. However, they are also expressed in inflammatory cells in rodent species where they promote the release of proinflammatory mediators.7 Because of this effect of A3ARs in inflammatory cells, it is theoretically possible that some of the actions of adenosine produced during ischemia may actually exacerbate ischemic injury. In support of this hypothesis, we have recently demonstrated that myocardial infarct size is smaller in mice in which the A3AR has been genetically disrupted14; that is, infarct size is smaller in mice lacking the expression of A3ARs in all tissues. Cerniway and colleagues26,27⇓ reported similar beneficial effects in A3AR knockout mice, which they initially ascribed to the absence of a proinflammatory action of A3ARs mediated through mast cell degranulation, 26 but have subsequently suggested may be the result of compensatory changes that developed in the knockout mice due to the chronic absence of A3ARs.27 The existence of two opposing effects of A3ARs (induction of protection as well as tissue injury via different mechanisms) could complicate the use of A3AR agonists to protect the ischemic myocardium. Targeted expression of the A3AR specifically in cardiac myocytes overcomes this potential problem and represents one approach to selectively enhance the beneficial actions of A3ARs.
With the advent of new approaches to transfer genes into the myocardium, it is possible that gene therapy will become feasible in patients with ischemic heart disease. Based on the results of the present study, the A3AR is one potential gene that should be considered for this purpose. It is apparent from the present results, however, that it will be necessary to develop strategies to control the level of expression of the A3AR in the heart to avoid unwanted side effects. At the present time, it is unclear whether it may be more prudent to target the A3AR versus the A1AR. Because the A3AR may have lower affinity for adenosine than the A1AR,28 overexpression of the A3AR may have less influence on the heart under basal conditions when adenosine levels are low. The A3AR transgenic mice described herein offer a unique tool to further study the function of A3ARs in the heart and to assess the therapeutic potential of overexpressing the A3AR for the treatment of ischemic heart disease.
This study was supported in part by National Institutes of Health Grants HL-60051 (J.A.A.), RR-11803 (J.A.A.), HL-43151 (R.B.), HL-55757 (R.B.), and HL-68088 (R.B.); and by American Heart Association National Center Grant 9630083N (J.A.A.). We gratefully acknowledge Mark E. Olah, PhD (Department of Molecular Pharmacology and Cellular Biophysics, University of Cincinnati, Cincinnati, Ohio) for providing 125I-AB-MECA for some of the studies and Jeffrey Robbins, PhD (Children’s Hospital Research Foundation, Cincinnati, Ohio) for providing the α-MyHC gene promoter.
This manuscript was sent to Stephen F. Vatner, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Original received October 19, 2001; resubmission received April 17, 2002; revised resubmission received June 19, 2002; accepted June 19, 2002.
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- ↵Takano H, Bolli R, Black RG Jr, Kodani E, Tang XL, Yang Z, Bhattacharya S, Auchampach JA. A1 or A3 adenosine receptors induce late preconditioning against infarction in conscious rabbits by different mechanisms. Circ Res. 2001; 88: 520–528.
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- ↵Cross HC, Murphy E, Black RG, Auchampach JA, Steenbergen CE. Overexpression of the cardiac A3 adenosine receptor (A3r) protects the ischemic heart. Circulation. 2000; 102: II-6.Abstract.
- ↵Dougherty C, Barucha J, Schofield PR, Jacobson KA, Liang BT. Cardiac myocytes rendered ischemia resistant by expressing the human adenosine A1 or A3 receptor. FASEB J. 1998; 12: 1785–1792.
- ↵Redfern CH, Degtyarev MY, Kwa AT, Salomonis N, Cotte N, Nanevicz T, Fidelman N, Desai K, Vranizan K, Lee EK, Coward P, Shah N, Warrington JA, Fishman GI, Bernstein D, Baker AJ, Conklin BR. Conditional expression of a Gi-coupled receptor causes ventricular conduction delay and a lethal cardiomyopathy. Proc Natl Acad Sci U S A. 2000; 97: 4826–4831.
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