Overexpression Of A1 Adenosine Receptors Research Articles

Sphingosine kinase 1 protects against renal ischemia–reperfusion injury in mice by sphingosine-1-phosphate1 receptor activation

The roles of sphingosine kinases SK1 and SK2 in ischemia-reperfusion injury have not been fully elucidated since studies have found beneficial effects of SK1 while others showed no role in this injury. To help resolve this, we used SK1 or SK2 knockout mice and confirmed that renal ischemia-reperfusion injury induced SK1, but not SK2, in the kidneys. Furthermore, knockout or pharmacological inhibition of SK1 increased injury after renal ischemia-reperfusion injury. In contrast, lack of SK2 conferred renal protection following injury. In addition, we used lentiviral gene delivery to selectively express enhanced green fluorescent protein (EGFP) or human SK1 coexpressed with EGFP (EGFP-huSK1) in the kidney. Mice with kidney-specific overexpression of EGFP-huSK1 had significantly improved renal function with lower plasma creatinine, renal necrosis, apoptosis, and inflammation. Moreover, EGFP-huSK1 overexpression in cultured human proximal tubule (HK-2) cells protected against peroxide-induced necrosis. Selective overexpression of EGFP-huSK1 led to increased HSP27 mRNA and protein expression in vivo and in vitro. Functional protection as well as induction of HSP27 with EGFP-huSK1 overexpression in vivo was blocked with sphingosine-1-phosphate-1 receptor(1) (S1P(1)) antagonism. Thus, our findings suggest that SK1 is renoprotective by S1P(1) activation and perhaps HSP27 induction. Kidney-specific expression of SK1 through lentiviral delivery may be a viable therapeutic option to attenuate renal ischemia-reperfusion injury.

Remodeling of inward rectifying K+ currents in rat atrial myocytes by overexpression of A1-adenosine receptors

In rat atrial myocytes GIRK (Kir3) channels can be activated by acetylcholine and adenosine via M(2) and A(1) receptors coupled to Pertussis-toxin-sensitive G proteins, such as M(2)R or A(1)R. Owing to the lower density of A(1)R, the amplitude of current activated by a saturating concentration (10μM) of Ado (I(K(Ado))) amounts to about 40% of maximum I(K(ACh)). Adenovirus-driven overexpression of A(1)R results in an increase in I(K(Ado)). In a fraction of A(1)R-overexpressing cells, both ACh and Ado failed to activate GIRK channels. These cells had a large constitutive Ba(2+)-sensitive inward rectifying background K(+) current, which was insensitive to the GIRK channel inhibitor tertiapin (200nM), suggesting this current component to be carried by I(K1) (Kir) channels. This effect of A(1)R overexpression was reduced by treatment (48h) with the A(1)R antagonist DPCPX. siRNA-mediated knockdown of Kir2.1, simultaneously with A(1)R overexpression, substantially reduced I(K1). The mechanisms underlying the upregulation of functional I(K1) channels involve activation of the phosphatidylinositol 3-kinase (Pi3K)/Akt (protein kinase B) pathway. Kir2.1 transcripts are not increased in myocytes overexpressing A(1)R. These data demonstrate that manipulation of the expression level of a G protein-coupled receptor has unpredictable effects on functional expression of proteins that are supposed to be unrelated to the pathway controlled by that GPCR.

Selective intrarenal human A1 adenosine receptor overexpression reduces acute liver and kidney injury after hepatic ischemia reperfusion in mice

Acute kidney injury (AKI) is frequent after liver ischemia reperfusion (IR) can potentiate liver injury and is often complicated by subsequent multiorgan dysfunction syndrome. AKI because of liver IR is characterized by early renal endothelial cell apoptosis and impaired vascular integrity with subsequent neutrophil infiltration, proximal tubule necrosis/inflammation, and filamentous (F) actin disintegration. We tested whether selective renal overexpression of human A1 adenosine receptors (huA1AR) protects against both liver and kidney injury sustained after liver IR. Mice were subjected to liver IR or to sham surgery 48 h after unilateral intrarenal injection of lentivirus encoding enhanced green fluorescent protein (EGFP) or EGFP-huA1AR. Intrarenal lentiviral gene delivery caused a robust transgene expression in the injected kidney without significant expression in the contralateral kidney or in the liver. Mice injected with EGFP-huA1AR lentivirus were protected against hepatic IR-induced liver and kidney injury with reduced necrosis, inflammation, and apoptosis, and better preserved F-actin and vascular permeability compared with mice injected with EGFP lentivirus. Importantly, we show that removing the EGFP-huA1AR lentivirus-injected kidney before hepatic ischemia abolished both renal and hepatic protection after liver IR showing that the overexpression of huA1AR in the injected kidney has a crucial role in protecting the kidney and liver after liver IR. Therefore, our findings show that protecting the kidney reduces liver IR injury and selective overexpression of cytoprotective A1ARs in the kidney leads to protection of both liver and kidney after hepatic IR.

Myocardial function following cold ischemic storage is improved by cardiac-specific overexpression of A1-adenosine receptors

Cold ischemic storage of hearts for transplantation is limited to 4-6 h, and therefore the development of strategies to extend preservation time may increase the donor pool of hearts. Overexpression of A1-adenosine receptors (A1AR) can protect hearts from acute ischemic injury, and the purpose of this study was to test the hypothesis that overexpression of A1AR will improve tolerance to longer periods of cold ischemic preservation. Hearts from 18 wild type and 16 transgenic mice with overexpression of A1AR (A1AR Trans) were isolated and perfused, and then subjected to 18 h of preservation in 5 degrees C University of Wisconsin solution followed by 2 h of reperfusion. Left ventricular end diastolic pressure and left ventricular developed pressure were measured as indices of ventricular function. Cell viability was assessed by determination of infarct size and myocardial cell apoptosis. A1AR Trans hearts showed improved function following 18 h of ischemia, as shown by lower end diastolic pressure (p < 0.05) and higher recovery of left ventricular developed pressure (p < 0.05) during reperfusion. A1AR Trans hearts had markedly reduced infarct size (p < 0.05) and decreased apoptosis (p < 0.05). Overexpression of cardiac A1AR imparts cardioprotection during long-term cold ischemic preservation.

Genetic Deletion of the A 1 Adenosine Receptor Limits Myocardial Ischemic Tolerance

Adenosine receptors may be important determinants of intrinsic ischemic tolerance. Genetically modified mice were used to examine effects of global A1 adenosine receptor (A1AR) knockout (KO) on function and ischemic tolerance in perfused mouse hearts. Baseline contractile function and heart rate were unaltered by A1AR KO, which was shown to abolish the negative chronotropic effects of 2-chloroadenosine (A1AR-mediated) without altering A2 adenosine receptor-mediated coronary dilation. Tolerance to 25 minutes global normothermic ischemia (followed by 45 minutes reperfusion) was significantly limited by A1AR KO, with impaired contractile recovery (reduced by 25%) and enhanced lactate dehydrogenase (LDH) efflux (increased by 100%). Functional effects of A1AR KO involved worsened systolic pressure development with little to no change in diastolic dysfunction. In contrast, cardiac specific A1AR overexpression enhanced ischemic tolerance with a primary action on diastolic dysfunction. Nonselective receptor agonism (10 micromol/L 2-chloroadenosine) protected wild-type and also A1AR KO hearts (albeit to a lesser extent), implicating protection via subtypes additional to A1ARs. However, A1AR KO abrogated effects of 2-chloroadenosine on ischemic contracture and diastolic dysfunction. These data are the first demonstrating global deletion of the A1AR limits intrinsic myocardial resistance to ischemia. Data indicate the function of intrinsically activated A1ARs appears primarily to be enhancement of postischemic contractility and limitation of cell death.

Altered signal transduction in cardiac ventricle overexpressing A(1)-adenosine receptors.

The aim of the present study was to assess the effects of A(1)-adenosine receptor (A1-AR) stimulation in ventricle of A(1)-adenosine receptor overexpressing mice (transgenic mice, TG). Effects of the A(1)-adenosine receptor agonist R-PIA ((-)-N(6)-phenylisopropyladenosine) on phosphorylation of phospholamban (PLB), Ca(2+) transients, Ca(2+) currents and cell shortening were studied in isolated ventricular cardiomyocytes. R-PIA alone did not affect contractility in isolated electrically stimulated cardiomyocytes from wild-type mice (WT) or TG. However, after pre-stimulation of beta-adrenoceptors by isoproterenol, R-PIA reduced contractility in cardiomyocytes from WT but increased contractility in TG. Under the same conditions, R-PIA reduced isoproterenol-stimulated currents through L-type Ca(2+) channels, Ca(2+) transients and phosphorylation of PLB in cardiomyocytes from WT. In contrast, R-PIA diminished phospholamban phosphorylation induced by isoproterenol but augmented isoproterenol-elevated currents through L-type Ca(2+) channels, and isoproterenol-heightened Ca(2+) transients in cardiomyocytes from TG. We suggest that A(1)-adenosine receptor overexpression reverses the interaction of beta-adrenergic and A(1)-adenosine receptor stimulation, at least in part. Hence, the receptor/effector coupling is dependent on receptor density in this model.

Adenosine

Adenosine has been recognized as a potentially important signaling molecule in the heart for nearly a half century. The original adenosine hypothesis proposed that production of adenosine by cardiac myocytes reflects the metabolic state of the myocardium and serves to regulate vasomotor tone in the coronary resistance vessels, thereby coupling blood flow to the energetic needs of the heart.1 There are two major pathways for adenosine production in the cardiac myocyte. The transmethylation pathway involves the hydrolysis of S -adenosylhomocysteine (SAH) by SAH hydrolase to l-homocysteine and adenosine. A second pathway, the hydrolysis of AMP by 5′-nucleotidase, predominates during ischemic or hypoxic conditions.2 According to the adenosine hypothesis, when the energy requirements of the heart increase, the increased rate of ATP hydrolysis would cause cytosolic free ADP levels to rise. In this situation, the myokinase reaction can utilize two molecules of ADP to produce one molecule each of ATP and AMP, the later being the substrate for 5′-nucleotidase to produce adenosine, which can enter the interstitial space to cause coronary vasodilation. Although this hypothesis for preserving the oxygen supply/demand relationship is attractive, it does not appear to operate during physiological conditions in the normal heart, since adenosine receptor inhibition does not decrease coronary blood flow or impair the increase of coronary flow during exercise.3 Implicit in the adenosine hypothesis is the assumption that during increased cardiac work, increases of cytosolic free [ADP] would be required to drive mitochondrial respiration and would serve to increase adenosine production. In fact, over a wide range of workloads, cytosolic free [ADP] remains at a stable low level; only at extreme workloads does ADP rise significantly in the normal heart.4 Although it has been difficult to demonstrate adenosine effects during normal physiological conditions, adenosine is clearly able to exert important effects …

Ischaemic tolerance in aged mouse myocardium: the role of adenosine and effects of A1 adenosine receptor overexpression.

The genesis of the ischaemia intolerant phenotype in aged myocardium is poorly understood. We tested the hypothesis that impaired adenosine-mediated protection contributes to ischaemic intolerance, and examined whether this is countered by A1 adenosine receptor (A1AR) overexpression. Responses to 20 min ischaemia and 45 min reperfusion were assessed in perfused hearts from young (2-4 months) and moderately aged (16-18 months) mice. Post-ischaemic contractility was impaired by ageing with elevated ventricular diastolic (32 +/- 2 vs. 18 +/- 2 mmHg in young) and reduced developed (37 +/- 3 vs. 83 +/- 6 mmHg in young) pressures. Lactate dehydrogenase (LDH) loss was exaggerated (27 +/- 2 vs. 16 +/- 2 IU g-1 in young) whereas the incidence of tachyarrhythmias was similar in young (15 +/- 1 %) and aged hearts (16 +/- 1 %). Functional analysis confirmed equipotent effects of 50 micro M adenosine at A1 and A2 receptors in young and aged hearts. Nonetheless, while 50 micro M adenosine improved diastolic (5 +/- 1 mmHg) and developed pressures (134 +/- 7 mmHg) and LDH loss (6 +/- 2 IU g-1) in young hearts, it did not alter these variables in the aged group. Adenosine did attenuate arrhythmogenesis for both ages (to ~10 %). In contrast to adenosine, 50 micro M diazoxide reduced ischaemic damage and arrhythmogenesis for both ages. Contractile and anti-necrotic effects of adenosine were limited by 100 micro M 5-hydroxydecanoate (5-HD) and 3 micro M chelerythrine. Anti-arrhythmic effects were limited by 5-HD but not chelerythrine. Non-selective (100 micro M 8-sulfophenyltheophylline) and A1-selective (150 nM 8-cyclopentyl-1,3-dipropylxanthine) adenosine receptor antagonism impaired ischaemic tolerance in young but not aged hearts. Quantitative real-time PCR and radioligand analysis indicated that impaired protection is unrelated to changes in A1AR mRNA transcription, or receptor density (~8 fmol mg-1 protein in both age groups). However, A1AR overexpression improved tolerance for both ages, restoring adenosine-mediated protection. These data reveal impaired protection via exogenous and endogenous adenosine contributes to ischaemic intolerance with ageing. This is independent of A1AR expression, and involves ineffective activation of a 5-HD-/diazoxide-sensitive process. The effects of A1AR overexpression indicate that the age-related failure in signalling can be overcome.

Mechanisms of cardiac protection with Overexpression of A1 adenosine receptors

AbstractMyocardial damage from ischemia/reperfusion injury can be minimized through endogenous protective mechanisms, including activation of A1 adenosine receptors. Transgenic mice with cardiac‐specific A1 adenosine receptor overexpression (A1AR Trans) have demonstrated functional and metabolic tolerance to in vitro and in vivo myocardial ischemia/reperfusion injury. The purpose of this investigation is to examine the role of p38 MAP kinase in A1 receptor‐mediated cardioprotection. Activation of p38 MAPK by A1 adenosine receptor stimulation was evaluated in neonatal rat cardiomyocytes subjected to simulated ischemia. A1 activation by cyclopentyladenosine decreased cell death and simultaneously enhanced p38 phosphorylation by simulated ischemia. The role of p38 MAP kinase activation in cardioprotection with A1 adenosine receptor overexpression was evaluated in isolated perfused hearts from A1AR Trans mice subjected to 20 min ischemia and 30 min reperfusion. Inhibition of p38 MAPK activity with 10 μM SB‐203580 prior to ischemia and during reperfusion decreased postischemic total functional recovery in A1AR Trans hearts from 54 ± 1 to 27±7% of maximal and increased final EDP from 10±3 mmHg to 34±8 mmHg. A1 adenosine receptor‐mediated activation of p38 MAP kinase in cardiomyocytes is involved in the protection of cells from ischemic death. Tolerance to ischemic injury by A1 receptor overexpression is dependent upon activation of p38 MAPK. These findings suggest that adenosine A1 mediated cardioprotection in transgenic mice involves activation of the p38 MAP kinase cascade. Drug Dev. Res. 58:439–446, 2003. © 2003 Wiley‐Liss, Inc.

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

We tested the hypothesis that myocardial ischemia-reperfusion (I/R)-induced apoptosis is attenuated in transgenic mice overexpressing cardiac A(1) 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 microM) 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 A(1) adenosine receptors involves attenuation of I/R-induced apoptosis that does not involve acute signaling through phosphoinositol-3-kinase.

A1 adenosine receptor overexpression decreases stunning from anoxia-reoxygenation: role of the mitochondrial K(ATP) channel.

Myocardial A1 adenosine receptor (A1AR) overexpression protects hearts from ischemia-reperfusion injury; however, the effects during anoxia are unknown. We evaluated responses to anoxia-reoxygenation in wild-type (WT) and transgenic (Trans) hearts with approximately 200-fold overexpression of A1ARs. Langendorff perfused hearts underwent 20 min anoxia followed by 30 min reoxygenation. In WT hearts peak diastolic contracture during anoxia was 45+/-3 mmHg, diastolic pressure remained elevated at 18+/-3 mmHg after reoxygenation, and developed pressure recovered to 52+/-4% of pre-anoxia. A1AR overexpression reduced hypoxic contracture to 29+/-4 mmHg, and improved recovery of diastolic pressure to 8+/-1 mmHg and developed pressure to 76+/-3% of pre-anoxia. Mitochondrial K(ATP) blockade with 100 microM 5-hydroxydecanoate (5-HD) increased hypoxic contracture to 73+/-6 mmHg in WT hearts, reduced post-hypoxic recoveries of both diastolic (40+/-5 mmHg) and developed pressures (33+/-3 %). In contrast, 5-HD had no effect on hypoxic contracture (24+/-8 mmHg), or post-hypoxic diastolic (10+/-2 mmHg) and developed pressures (74+/-3%) in Trans hearts. In summary, (i) A1AR overexpression improves myocardial tolerance to anoxia-reoxygenation, (ii) intrinsic mitochondrial K(ATP) channel activation decreases hypoxic contracture and improves functional recovery in wild-type hearts, and (iii) mitochondrial K(ATP) channels do not appear to play a major role in the functional protection from anoxia afforded by A1AR overexpression.

Cardiac overexpression of A1-adenosine receptor protects intact mice against myocardial infarction.

Previous studies have shown that high-level (300-fold normal) cardiac overexpression of A1-adenosine receptors (A1-ARs) in transgenic (TG) mice protects isolated hearts against ischemia-reperfusion injury. However, this high level of overexpression is associated with bradycardia and increased incidence of arrhythmia during ischemia in intact mice, which interfered with studies to determine whether this line of TG mice might also be protected against myocardial infarction (MI) in vivo. For these studies, we therefore selected a line of TG mice that overexpresses the A1-AR at more moderate levels (30-fold normal), which affords cardioprotection in the isolated heart while minimizing bradycardia and arrhythmia during ischemia in intact mice. Wild-type (WT; n = 10) and moderate-level A1-AR TG (n = 10) mice underwent 45 min of left anterior descending coronary artery occlusion, followed by 24-h reperfusion. Infarct size and region at risk were determined by triphenyltetrazolium chloride and phthalo blue staining, respectively. Infarct size (% region at risk) in WT mice was 52 +/- 3%, whereas overexpression of A1-ARs in the TG mice markedly reduced infarct size to 31 +/- 3% (P < 0.05). Furthermore, contractile function (left ventricular ejection fraction) as determined by cardiac magnetic resonance imaging 24 h after MI was better preserved in TG vs. WT mice. Cardiac overexpression of A1-ARs reduces infarct size by 40% and preserves cardiac function in intact mice after MI.

Functional studies in atrium overexpressing A1-adenosine receptors.

1. Adenosine and the A1-adenosine receptor agonist R-PIA, exerted a negative inotropic effect in isolated, electrically driven left atria of wild-type mice. 2. In left atria of mice overexpressing the A1-adenosine receptor, adenosine and R-PIA exerted a positive inotropic effect. 3. The positive inotropic effect of adenosine and R-PIA in transgenic atria could be blocked by the A1-adenosine receptor antagonist DPCPX. 4. In the presence of isoprenaline, adenosine exerted a negative inotropic effect in wild-type atria but a positive inotropic effect in atria from A1-adenosine receptor overexpressing mice. 5. The rate of beating in right atria was lower in mice overexpressing A1-adenosine receptors compared with wild-type. 6. Adenosine exerted comparable negative chronotropic effects in right atria from both A1-adenosine receptor overexpressing and wild-type mice. 7. A1-adenosine receptor overexpression in the mouse heart can reverse the inotropic but not the chronotropic effects of adenosine, implying different receptor-effector coupling mechanisms.

Functional genomics of transgenic overexpression of A1 adenosine receptors in the heart

The advent of transgenic techniques provides a unique opportunity to study adenosine receptor-mediated cardioprotection. Transgenic A 1 receptor overexpression increases myocardial protection without impairing intrinsic function, but at the expense of resting bradycardia and a blunted response to catecholamine stimulation. Thus, we investigated whether an optimal degree of A 1 receptor overexpression exists whereby hearts would be protected from ischemic injury with minimal sequelae. Hearts from two transgenic lines were evaluated (high-level expressors, HL, and low-level expressors, LL) and compared to controls. Both transgenic lines showed enhanced myocardial protection as measured by postischemic recovery of developed tension (HL 49 ± 3% of baseline, LL 45 ± 3%, and control 31 ± 3%; P < 0.05). Resting heart rates in conscious restrained mice were lowest in HL (620 ± 14 bpm), intermediate in LL (681 ± 11 bpm), and highest in control (713 ± 8 bpm), P < 0.05, while systolic blood pressure was similar in all groups. The inotropic response to isoproterenol was comparably blunted in HL and LL hearts (+dP/dt increased 33 ± 4% and 39 ± 3%, respectively) compared to control (58 ± 7%), P < 0.05. β-adrenergic receptor density was upregulated only in the HL hearts (90 ± 12 fmol/mg) compared to LL and control (41 ± 9 and 32 ± 5, respectively, P < 0.05). In summary, both HL and LL hearts afforded cardioprotection from ischemia, with LL hearts exhibiting less bradycardia and less β-adrenergic cross-regulation. While the data suggest that genetic manipulation of receptor systems may provide a powerful tool to improve myocardial tolerance to injury, they also highlight the need for careful screening for effects on the integrated physiology of the system.

Transgenic A1 adenosine receptor overexpression markedly improves myocardial energy state during ischemia-reperfusion.

A1 adenosine (A1AR) activation may reduce ischemia-reperfusion injury. Metabolic and functional responses to 30 min global normothermic ischemia and 20 min reperfusion were compared in wild-type and transgenic mouse hearts with approximately 100-fold overexpression of coupled cardiac A1ARs. 31P-NMR spectroscopy revealed that ATP was better preserved in transgenic v wild-type hearts: 53 +/- 11% of preischemic ATP remained after ischemia in transgenic hearts v only 4 +/- 4% in wild-type hearts. However, recovery of ATP after reperfusion was similar in transgenic (46 +/- 5%) and wild-type hearts (37 +/- 12%). Reductions in phosphocreatine (PCr) and cytosolic pH during ischemia were similar in both groups. However, recovery of PCR on reperfusion was higher in transgenic (67 +/- 8%) v wild-type hearts (36 +/- 8%), and recovery of pH was greater in transgenic (pH = 7.11 +/- 0.05) v wild-type hearts (pH = 6.90 +/- 0.02). Bioenergetic state ([ATP]/[ADP].[Pi]) was higher in transgenic v wild-type hearts during ischemia-reperfusion. Time to ischemic contracture was prolonged in transgenic (13.6 +/- 0.8 min) v wild-type hearts (10.4 +/- 0.3 min). Degree of contracture was lower and recovery of function in reperfusion higher in transgenic v wild-type hearts. In conclusion, A1AR overexpression reduces ATP loss and improves bioenergetic state during severe ischemic insult and reperfusion. These changes may contribute to improved functional tolerance.

Myocardial Protection from Ischemic Preconditioning is Mimicked by Transgenic Overexpression of A1 Adenosine Receptors ♦ 127

“Preconditioning” the heart with brief periods of ischemia protects it from sustained ischemia such as can occur during surgical correction of congenital heart disease. One possible mechanism of this protection involves the activation of A1 adenosine receptors. To investigate the role of A1 adenosine receptors in ischemic preconditioning, wild-type and transgenic mouse hearts with increased A1 receptors were studied. The effect of preconditioning on recovery from sustained ischemia was examined in isolated Langendorff hearts in each group. Myocardial injury was assessed by postischemic functional recovery (% of baseline developed tension,% DT) and quantitation of enzyme markers of cell death in coronary effluent (creatine kinase, CK and lactate dehydrogenase, LDH; u/L/gm for both). Transgenic overexpression of A1 receptors resulted in greater recovery of developed tension (45% vs 26% p<0.05, ANOVA) and nearly undetectable levels of CK and LDH compared to wild-type (table). Preconditioning wild-type hearts improved recovery of developed tension similar to A1 receptor overexpression (38% vs 45%, n.s.) and also resulted in very low levels of CK and LDH. Interestingly, preconditioning in transgenic hearts caused no further improvement of function (39% vs 45%, n.s.). In summary, both preconditioning and transgenic overexpression of A1 adenosine receptors result in similar improvement of functional recovery and decreased cell death following ischemia. The inability of preconditioning to further enhance the functional recovery in transgenic mice suggests that hearts with A1 adenosine receptor overexpression may already be preconditioned.

Myocardial Function in the Working Mouse Heart Overexpressing Cardiac A1Adenosine Receptors

Adenosine, acting via A1receptors, modulates heart rate and contractility, and provides myocardial protection during times of stress. A transgenic model of cardiac A1overexpression was produced and it demonstrated cardiac protection from ischemia. Since A1receptor stimulation can inhibit contractility under some conditions, the present study was undertaken to determine the effects of transgenic A1overexpression on intrinsic contractility and the response to catecholamine stimulation. Isolated working mouse hearts were subjected to volume- and pressure-loading protocols to assess intrinsic contractility, and isoproterenol infusions to assess catecholamine response. Basal heart rates were lower in transgenic (Trans) hearts than controls (Ctrl), but with pacing baseline cardiac function and contractility (as measured by +dP/dt) were similar. Volume and pressure loading of Ctrl and Trans hearts were also similar along the entire range tested. No differences were seen in the sensitivity to isoproterenol infusion, but at maximal doses there was a decrease in maximum +dP/dtin Trans hearts compared to Ctrl (maximum +dP/dt152±6% baseline for Ctrl, 131±2% baseline for Trans,P<0.05). In summary, overexpression of A1receptors does not produce untoward effects on ventricular function or sensitivity to catecholamine stimulation, but does dampen the contractile response at high doses of catecholamines. These data suggest that even with 1000-fold overexpression of A1adenosine receptors, adenosine plays little or no role in regulating intrinsic myocardial contractility in the sympathectomized isolated working heart, only modulating contractility as the heart becomes stressed during exposure to higher catecholamine levels.

Transgenic A1 adenosine receptor overexpression increases myocardial resistance to ischemia.

Activation of myocardial A1 adenosine receptors (A1AR) protects the heart from ischemic injury. In this study transgenic mice were created using the cardiac-specific alpha-myosin heavy chain promoter and rat A1AR cDNA. Heart membranes from two transgene positive lines displayed approximately 1,000-fold overexpression of A1AR (6,574 +/- 965 and 10,691 +/- 1,002 fmol per mg of protein vs. 8 +/- 5 fmol per mg of protein in control hearts). Compared with control hearts, transgenic Langendorff-perfused hearts had a significantly lower intrinsic heart rate (248 beats per min vs. 318 beats per min, P < 0. 05), lower developed tension (1.2 g vs. 1.6 g, P < 0.05), and similar coronary resistance. The difference in developed tension was eliminated by pacing. Injury of control hearts during global ischemia, indexed by time-to-ischemic contracture, was accelerated by blocking adenosine receptors with 50 microM 8-(p-sulfophenyl) theophylline but was unaffected by addition of 20 nM N6-cyclopentyladenosine, an A1AR agonist. Thus A1ARs in ischemic myocardium are presumably saturated by endogenous adenosine. Overexpressing myocardial A1ARs increased time-to-ischemic contracture and improved functional recovery during reperfusion. The data indicate that A1AR activation by endogenous adenosine affords protection during ischemia, but that the response is limited by A1AR number in murine myocardium. Overexpression of A1AR affords additional protection. These data support the concept that genetic manipulation of A1AR expression may improve myocardial tolerance to ischemia.