Regional Desensitization of -Adrenergic Receptor Signaling in Swine With Chronic Hibernating Myocardium
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循环研究杂志 2005年第10期
the Veterans Affairs Western New York Health Care System (V.S.I., J.M.C.), Buffalo
the Departments of Medicine (V.S.I., J.M.C.) and Physiology & Biophysics (J.M.C.), University at Buffalo, NY.
Abstract
Contractile reserve during submaximal -adrenergic stimulation is attenuated in patients and swine with hibernating myocardium. We tested the hypothesis that this arises as a regional adaptive response in -adrenergic adenylyl cyclase coupling. Pigs (n=8) were studied 3 months after instrumentation with a left anterior descending artery (LAD) stenosis when flow (LAD, 0.7±0.2 versus 1.2±0.1 mL/min per gram in normal remote; P<0.05) and wall thickening (LAD, 5.5±3.2% versus 40.0±5.5% in remote; P<0.05) were reduced in the absence of infarction. Whereas basal cAMP production was normal (LAD, 87±18 versus 91±19 pmol/mg per minute; P=NS), responses to isoproterenol were blunted (LAD, 83±6 versus 146±25 pmol/mg per minute in remote; P<0.05). -receptor density and subtype were unchanged, but there was a reduction in the number of high-affinity binding sites (LAD, 40±4% versus 53±7% in normal remote; P<0.05). The Gi2/Gs ratio increased (LAD, 1.8±0.3 versus 0.99±0.3 in remote myocardium; P<0.05), although GppNHp-stimulated cAMP production was equivocally reduced. Forskolin responses were unchanged and similar to shams. These data indicate regional attenuation of -receptor adenylyl cyclase signaling in hibernating myocardium. This blunts the local contractile response to -adrenergic stimulation and may serve to protect against a myocardial supply/demand imbalance when external determinants of myocardial workload increase during sympathetic activation.
Key Words: -receptors hibernating myocardium chronic ischemic heart disease adenylyl cyclase
Introduction
Myocardial viability is an important determinant of prognosis in ischemic heart disease and is frequently identified by an increase in regional function in response to -adrenergic stimulation.1eC3 Unfortunately, a significant number of patients have limited contractile reserve when viability is present as assessed by nuclear techniques. The absence of contractile reserve appears to be particularly common in the subset of patients with hibernating myocardium where resting perfusion and function are both reduced, as compared with those with chronic stunning, where function is reduced and resting perfusion is normal.4eC6 We recently used a porcine model that progresses from stunned to chronic hibernating myocardium7 to demonstrate a similar attenuation of regional function during graded -adrenergic stimulation.8 Although the increase in flow was attenuated in the presence of a critical LAD stenosis, intrinsic adaptive responses in hibernating myocardium prevented the development of ischemia, as reflected by increased subendocardial function in the absence of lactate production during submaximal -adrenergic stimulation.9 These results suggest that hibernating myocardium operates at a lower point of the supply/demand relation than normal myocardium to limit an energetic imbalance during increases in external myocardial workload. The adaptations responsible for the attenuated contractile response to -adrenergic stimulation have not been established.
One potential adaptation that could explain the attenuated contractile reserve is a regional reduction in -adrenergic responsiveness arising from repetitive reversible ischemia. Although no previous studies have evaluated -receptor signaling in chronic hibernating myocardium, stunned myocardium has been reported to have increased -receptor density, little change in -receptoreCmediated cAMP production, and an accentuated contractile response.10,11 Although this is similar to the denervation hypersensitivity seen following transmural myocardial infarction, these findings have not been reproduced by others.12 Importantly, Shan et al actually demonstrated a reduction in -adrenergic surface density in needle biopsies from humans with hibernating myocardium.13 Such effects would be predicted to lead to an attenuated rather than accentuated contractile response to sympathetic stimulation and are consistent with in vivo observations in swine with hibernating myocardium.8,9,14
We, therefore, hypothesized that the attenuated metabolic and functional response to submaximal -adrenergic stimulation in pigs with hibernating myocardium arises from a regional downregulation in -adrenergic adenylyl cyclase coupling. To test this, we assessed in vitro -receptor density and affinity, stimulated cAMP production, and changes in regional G-protein expression in swine. The results demonstrate regional attenuation of -adrenergic signaling in hibernating myocardium that provides an intrinsic adaptation to limit the development of regional ischemia during increases in external workload.
Materials and Methods
Experimental procedures and protocols conformed to institutional guidelines for the care and use of animals in research. Hibernating myocardium was produced in pigs as previously described.7 Tissue for the present study was obtained from a series of animals used to assess in vivo myocardial norepinephrine uptake.15 Using sterile technique, juvenile pigs (n=8) were instrumented with a 1.5- to 2.0-mm fixed-diameter Delrin stenosis placed on the proximal left anterior descending artery (LAD). Three months after instrumentation, physiological studies were performed in the closed-chest sedated state (propofol 2 to 5 mg/kg per minute, IV supplemented with Telazol/xylazine).
We placed a 6F introducer into the brachial artery and inserted a 5F end-hole micromanometer (Millar Inc, Houston, Tex) into the left ventricle. Aortic pressure was monitored via the side port of the introducer. Blood flow measurements were determined at rest and during pharmacological vasodilation with adenosine (0.9 mg/kg per minute IV with phenylephrine coinfused to prevent hypotension) using fluorescent microspheres.8 Echocardiography was used to assess regional wall thickening in the hibernating LAD and normally perfused remote regions.8 End diastole was defined at the peak of the R wave and end systole was evaluated at minimum left ventricular (LV) chamber volume. The animals were allowed to recover and tissue sampling was performed at least 3 days after pharmacological stimulation.15 Tissue from the center of the hibernating region (adjacent to the LAD) was compared with normal remote LV myocardial samples taken adjacent to the posterior descending artery. Four additional sham animals were used to exclude changes in remote myocardium. Light microscopy and point counting were performed to quantify connective tissue.7
Membrane Preparation
Flash frozen (eC80°C) subendocardial samples were powdered in a stainless-steel mortar and pestle cooled with liquid nitrogen and homogenized using a polytron (setting, 4 to 5) in a buffer containing 50 mmol/L Tris (pH 7.4), 20% sucrose, 2 mmol/L EGTA, 10 e/mL Leupeptin, 10 e/mL pepstatin, 10 e/mL aprotinin, and 5 e/mL phenylmethylsulfonyl fluoride. After homogenates were centrifuged (10 minutes; 2500g), we centrifuged the supernatant (20 minutes; 48 000g). Pellets were resuspended in 50 mmol/L Tris or 30 mmol/L HEPES (pH 7.4) and frozen at eC80°C for subsequent use in radioligand-binding experiments, adenylyl cyclase assays, and Western blot analysis. Membrane protein concentrations were determined in triplicate and yields were similar in LAD and remote regions (9.9±0.5 mg/g versus 10±0.5 mg/g; P=NS).
Adenylyl Cyclase Activity
Adenylyl cyclase activity was determined using a modification of the method by Salomon et al.16 Assays were performed in 100 e蘈 of solution containing 15 mmol/L HEPES (pH 7.4), 5 mmol/L MgCl2, 0.4 mmol/L ATP, 5 mmol/L creatine phosphate, 0.5 mmol/L EGTA, 50 mmol/L NaCl, 1 mmol/L cAMP, 60 U/mL creatine phosphokinase, 1 mmol/L 3-iso-butyl-2-methylxanthine, and 2 e藽i [32P]ATP. Reactions were initiated by adding 100 e of membrane protein and incubated at 37°C for 20 minutes. The reaction was terminated by adding 100 e蘈 of stopping solution (2% sodium dodecyl sulfate, 40 mmol/L ATP, and 1.4 mmol/L cAMP). We added [3H] (5000 counts per minute) to each reaction for estimation of column recovery. The [32P]cAMP produced was separated from [32P]ATP by sequential Dowex and alumina chromatography. Column recovery was 85% to 95%. Adenylyl cyclase activity was determined under basal conditions and after stimulation with isoproterenol (10eC4 mol/L in the presence of 0.1 mmol/L GTP), forskolin (10eC4 mol/L), and GppNHp (10eC4 mol/L). In preliminary experiments, we performed dose-response curves with each of these agents to determine the concentration eliciting maximal adenylyl cyclase cAMP production for use in subsequent studies. Data represent total cAMP production. Baseline cAMP production before stimulation was not different from the basal state (P>0.05, ANOVA).
Western Blotting
Regional expression of G proteins (25 e/lane) and G-protein receptor kinase (GRK) (40 e/lane) was determined on membrane preparations as previously described.17 Paired experimental and normal samples were run simultaneously to minimize variability. Equal loading and transfer were confirmed in preliminary experiments (Coomassie blue and Ponceau S staining). Nitrocellulose membranes were incubated with antibodies (Santa Cruz Biotechnology Inc) to G proteins (Gs [SC-823], 1:1000; Gi2 [SC-391], 1:1000 dilution) and GRK-2 and GRK-5 (GRK-2 [SC-562] and GRK-5 [SC-565]; each at 1:500 dilution) overnight at 4°C. They were subsequently incubated for 1 hour with 0.1% TBS containing horseradish peroxidaseeCconjugated anti-rabbit IgG at 1:10 000 dilutions, visualized with the Supersignal West Peco chemiluminescent substrate (Pierce, Ill), and quantified with a laser densitometer (Bio-Rad Inc).
Radioligand Binding Experiments
-Adrenergic receptors were quantified using 125I-iodocyanopindolol (5 to 450 pmol/L) in saturation isotherm experiments conducted in membrane preparations as previously described.18 In brief, 100 e of membrane protein prepared as described above was suspended in binding buffer containing 250 mmol/L HEPES, 20 mmol/L MgCl2, and 0.2% BSA. The membranes were incubated with increasing concentrations of 125I-iodocyanopindolol for 1 hour at 37°C in a water bath. Nonspecific binding was defined as binding in the presence of 10 eol/L propranolol. All reactions were performed in triplicate and specific binding was >80%. The reactions were terminated by rapid filtration using a Brandel M-24R cell harvester (Gaithersburg, Md) through Whatman GF/C filters followed by 4 washes with 4 mL of wash buffer (20 mmol/L Tris-HCl [pH 7.4], 2 mmol/L MgCl2). The filters were transferred to polystyrene tubes and counted in a gamma counter for 1 minute. The antagonist binding data were analyzed using the Graph Pad Prism program. The proportion of receptors in high-affinity and low-affinity states were determined in competition-binding experiments by incubating 100 pmol/L 125I-iodocyanopindolol with sixteen increasing concentrations of isoproterenol (10eC11 to 10eC4 mol/L) in the absence of guanine nucleotides. The binding assays were performed in the buffer described above with 3-hour incubation at 30°C. All reactions were performed in duplicate, with nonspecific binding assessed in the presence of 0.1 mmol/L isoproterenol. The displacement curves were fit to 1-site or 2-site binding models using the Graph Pad Prism 4 curve-fitting program. A 1-way ANOVA compared 2- and 1-site fits. Finally, proportions of 1 and 2 receptors were determined in competition-binding experiments using 16 different concentrations of CGP20712A (10eC11 to 10eC4 mol/L), a selective 1 antagonist.
Statistical Analysis
Data are reported as the mean±SEM. After an ANOVA, post hoc analysis was performed with paired 2-tailed t tests. Unpaired t tests were used for sham comparisons. A value of P<0.05 was significant.
Results
All pigs were in good health at the time of study and 2eC3-5-triphenyl tetrazolium chloride staining showed no evidence of acute or healed infarction. Angiography demonstrated total occlusion of the LAD, which filled by collaterals arising from both left and right coronary arteries. There was anterior hypokinesis consistent with viable dysfunctional myocardium. Although point counting demonstrated an increase in interstitial connective tissue typical of hibernating myocardium (7.3±1.4% versus 3.6±0.5% in remote regions; P<0.05), >90% of both regions was comprised of myocytes.
Under resting conditions, heart rate averaged 94±7 bpm, LV systolic pressure averaged 147±7 mm Hg, and LV end-diastolic pressure averaged 23±2 mm Hg. Measurements of resting flow and wall thickening confirmed the presence of hibernating myocardium. In the subendocardium (Figure 1A), flow was reduced at rest (LAD, 0.75±0.14 versus 1.19±0.14 mL/min per gram in normal remote myocardium; P<0.05). Like the hypokinesis demonstrated on ventriculography, LAD wall thickening was also significantly reduced (Figure 1B; LAD, 5.5±3.2% versus 40.0±5.5% in remote; P<0.05). Following adenosine, subendocardial flow in the hibernating region was critically impaired (0.7±0.2 mL/min per gram) and unable to increase above resting values, whereas flow in the remote region increased 4 times (4.7±0.8 mL/min per gram; P<0.05, LAD versus remote). Thus, subendocardial samples had physiological features of hibernating myocardium in the absence of infarction.
Adenylyl Cyclase Activity
Figure 2 summarizes results of the adenylyl cyclase activity assays. Total cAMP accumulation was similar under basal conditions (LAD, 87±18 versus 91±19 pmol/mg per minute in remote). Stimulation with isoproterenol (10eC4 mol/L) resulted in an attenuated cAMP accumulation in hibernating myocardium (LAD, 83±6 versus 146±25 pmol/mg per minute in remote; P<0.05). Stimulation of adenylyl cyclase with GppNHp (10eC4 mol/L) was also attenuated in hibernating myocardium, but the difference was of borderline significance (LAD, 202±37 versus 277±64 pmol/mg per minute in remote; P=0.11). Forskolin-stimulated responses (10eC4 mol/L) were not different (LAD, 276±39 versus 299±43 pmol/mg per minute in remote; P=NS). Thus, there was a regional attenuation of -mediated stimulation of adenylyl cyclase and normal stimulation with forskolin in chronic hibernating myocardium.
In sham instrumented pigs, basal (LAD, 103±19 versus 97±22 pmol/mg per minute in remote; P=NS), isoproterenol-stimulated (LAD, 156±14 versus 152±18 pmol/mg per minute in remote; P=NS), and forskolin-stimulated (LAD, 348±31 versus 335±28 pmol/mg per minute in remote; P=NS) cAMP were similar. Remote myocardial values in shams were not significantly different from remote values in animals with hibernating myocardium.
G-Protein and GRK Immunoblotting
Analysis of G protein levels (Figure 3) revealed an increase in Gi2 levels in hibernating myocardium (LAD, 11.2±2.2 versus 7.2±1.2 densitometric units in remote; P<0.05). There was a trend toward reduced membrane-associated levels of Gs, which did not reach statistical significance (LAD, 7.7±1.2 versus 12.2±3.3 densitometric units in remote; P=0.15). There were no regional differences in Gi2 or Gs proteins in sham animals. Thus, the Gi2/Gs ratio in hibernating regions was significantly increased (LAD, 1.84±0.3 versus 0.9±0.3 in remote myocardium; P<0.005). GRK-2 protein levels were similar in remote and hibernating tissue, whereas GRK-5 protein showed a trend toward increasing in hibernating myocardium (LAD, 7.8±1.2 versus 6.8±1.1 densitometric units in remote; P=0.15).
Radioligand Binding
The Table summarizes results of radioligand-binding experiments. Scatchard analyses r2 values were 0.96±0.01. There was no difference in total -receptoreCbinding sites using 125I-iodocyanopindolol in hibernating versus remote myocardium. Displacement experiments using 125I-iodocyanopindolol to assess changes in high- and low-affinity receptors (Figure 4) consistently fit best to a 2-site model (ANOVA, P<0.05) and were shifted to the right in hibernating myocardium. This reflected a reduction in the number of high-affinity binding sites in hibernating myocardium (40±4% versus 53±7%; P<0.05). Competition-binding experiments done in the presence of increasing concentrations of CGP20712A (Figure 5), a selective 1 antagonist, did not reveal any differences in the proportion of 1 and 2 receptor subtypes (Table).
Discussion
There are several new and important findings from our study. First, there is a regional attenuation of -adrenergiceCmediated cAMP production in subendocardial samples from hibernating myocardium that occurs in the absence of infarction. This was not attributable to alterations in adenylyl cyclase activity because direct stimulation with forskolin was not affected. Second, whereas there was no alteration in total -receptor density in vitro, competition-binding curves demonstrated a reduction in -receptor affinity. This was associated with a regional reduction in the number of high-affinity binding sites and increase in Gi2 proteins. Collectively, our results support the notion that an adaptive downregulation of -adrenergic responsiveness contributes to attenuating increases in regional metabolic demand during increases in external workload from sympathetic activation.
Effects of Reversible Ischemia on -Adrenergic Signaling
The effects of ischemia on -receptor signaling have been studied in a variety of models of reversible transmural ischemia leading to stunned myocardium. Although the specific alterations depend on the model used, as well as the duration of ischemia, most of the available data have demonstrated no change or evidence of accentuated -receptor adenylyl cyclase coupling. For example, Sato et al found wall thickening after isoproterenol increased more in stunned myocardium than in control, a finding potentially consistent with -adrenergic hypersensitivity following ischemia.11 Whereas this was associated with a small increase in -receptor density, isoproterenol-stimulated cAMP production was unchanged. In chronically stunned myocardium, in vivo -adrenergic stimulation normalized regional function, although effects on -adrenergic adenylyl cyclase production were not reported.10 In a model using multiple brief episodes of ischemia, Boraso et al found no changes in cAMP, -receptor density, affinity, or G-protein levels.19 Likewise, prolonged moderate ischemia or short-term hibernation in swine was associated with contractile reserve to dobutamine and unchanged -receptor levels, with no evidence of denervation hypersensitivity.12 Thus, following reversible ischemia, the functional response to transient -adrenergic stimulation is either normal or accentuated and biochemical evidence of alterations in cellular -receptor signaling is limited.
Hammond et al studied -receptor signaling in pigs subjected to repetitive ischemia from an ameroid constrictor placed around the left circumflex artery.20 Although demand-induced ischemia was produced in their model, the physiological significance of the stenosis was not sufficient to cause chronic reductions in resting flow or function. Thus, these investigators were able to evaluate the effects of repetitive ischemia on -adrenergic adenylyl cyclase coupling independently of the progression to stunned or hibernating myocardium. Like the present study, they reported a regional reduction in -receptor affinity with a shift to a low-affinity state in dysfunctional myocardium. Although these changes were accompanied by a reduction in -receptor number, there were no effects on stimulated adenylyl cyclase activity. These paradoxical findings were explained by a reciprocal reduction in Gi2 and an increase in Gs compensating for the alteration in -receptor number and affinity.
Our results in hibernating myocardium differ from those reported in myocardium subjected to reversible ischemia with or without stunning in several respects. We found that cAMP production in response to isoproterenol stimulation was regionally reduced in chronic hibernating myocardium. This supports previous in vivo functional observations in this model demonstrating an attenuated contractile response to -adrenergic stimulation with epinephrine when assessed using regional wall motion by ventriculography,7 regional wall-thickening measurements from echocardiography,8 and subendocardial segment length shortening.9,14 In each circumstance, -adrenergic stimulation improved but did not normalize myocardial function in hibernating myocardium. Radioligand-binding data in the presence of 125I-cyanopindolol demonstrated that Bmax (total receptor binding sites) was similar in remote and hibernating regions, indicating that total -adrenergic receptor density was normal. Competition binding revealed a loss of high-affinity receptor binding sites in hibernating myocardium, which at least partly explained the reduced ability of -adrenergic stimulation to increase cAMP in response to isoproterenol. The mechanism for the loss of high-affinity sites is most likely an uncoupling of the receptor from the G-protein. While speculative, phosphorylation of the receptor by protein kinases, including G-protein coupled receptor kinases, could cause uncoupling of the receptoreCG-protein complex.21eC23 Although the increase in GRK-5 levels was not significant, and GRK-2 protein levels were unchanged, it is plausible that their activities are significantly enhanced in hibernating myocardium and will require further studies.
Role of Altered G-Protein Expression on Adenylyl Cyclase in Hibernating Myocardium
The differences in -adrenergic signaling caused by chronic ischemia and hibernating myocardium lead to opposing effects of these states on G-protein expression. We found reciprocal alterations in the levels of G proteins in hibernating myocardium with the inhibitory subunit Gi2 increasing and the stimulatory subunit Gs decreasing. These are the exact opposite of the changes reported in myocardium with normal resting function subjected to chronic repetitive ischemia by Hammond et al.20 Coupled with the differences in -adrenergiceCmediated cAMP production, the results support the notion that hibernating myocardium reflects a fundamentally different phase of the chronic adaptive response to repetitive ischemia.
The functional importance of alterations in G-protein levels in hibernating myocardium is unclear because relative protein changes were greater than the reduction in GppNHp-stimulated adenylyl cyclase activity. This could reflect the possibility that G-protein changes have no functional impact because of the molar excess of G-proteins as compared with receptors and adenylyl cyclase molecules.24 Alternatively, it could reflect differential activation of the individual G-proteins or compartmentalization of a pool of G-proteins that, along with receptors and adenylyl cyclase molecules, colocalize in microdomains of the cell membrane lined by caveolar proteins.25 Thus, the stoichiometric excess of G-proteins in the whole cell might not represent the pool of G-proteins available to the receptors in caveolar microdomains. Finally, it is conceivable that there is an effect on the dose-response relation to GppNHp that could be brought out at submaximal concentrations, which we did not study. Further studies will be required to determine the functional impact of chronic alterations in G-protein expression because these changes may impact on other adaptive responses in hibernating myocardium.
Relation of Postsynaptic Reductions in -Adrenergic Receptor Signaling to Cellular Hypertrophy and Reductions in Presynaptic Norepinephrine Uptake
Although regional, our findings in hibernating myocardium are reminiscent of those reported in models of heart failure, where there are global reductions in -receptor signaling in the presence of systemic neurohormonal activation and reduced presynaptic norepinephrine uptake.26 In some heart failure models, these changes are accompanied by global reductions in the expression of the sarcoplasmic reticulum calcium-uptake proteins, phospholamban, and the sarcoplasmic reticulum Ca2+ATPase, which is seen regionally in the absence of neurohormonal activation and heart failure in hibernating myocardium.17 Whereas global cellular hypertrophy occurs in the failing heart, hibernating myocardium is associated with regional hypertrophy secondary to low but chronic rates of apoptosis.27 A number of laboratories have demonstrated altered -adrenergic adenylyl cyclase coupling in models of cellular hypertrophy that are not associated with decompensation or neurohormonal activation.28 Thus, it is conceivable that the alterations in -adrenergic affinity and attenuated cAMP production reflect a common response of the hypertrophied cardiac myocyte.
Although the presence of normal -adrenergic signaling in remote regions from the same heart excludes systemic neurohormonal activation as a mechanism in the present study, compartmental increases in norepinephrine are an alternative mechanism leading to the regional downregulation in -receptor signaling. We have previously demonstrated that regional norepinephrine uptake is moderately reduced in hibernating myocardium, as assessed by the uptake of 125IeCmeta-iodo-benzylguanidine ex vivo15 and imaging 11C-hydroxyephedrine uptake using positron emission tomography in vivo.29 These regional changes may reflect a loss of sympathetic nerves or a functional impairment in norepinephrine uptake. If reduced norepinephrine uptake was indicative of partial sympathetic denervation, it would be predicted to result in accentuated functional responses and increased -adrenergiceCmediated cAMP production. Although such effects have been seen several weeks after surgical sympathetic denervation in animals,30 the blunted cAMP production and functional responses during -adrenergic stimulation argue against this mechanism in hibernating myocardium.
Our results support a fundamentally different postsynaptic response than that seen in the denervated heart. The most likely explanation is that alterations in 125IeCmeta-iodo-benzylguanidine and 11C-hydroxyephedrine reflect chronic functional abnormalities in the presynaptic norepinephrine uptake mechanism rather than denervation. In this circumstance, locally increased norepinephrine overflow during sympathetic activation would be predicted to lead to a regional downregulation in -adrenergic receptor function rather than -adrenergic hypersensitivity. A similar mechanism has been hypothesized to contribute to chamber-specific -receptor downregulation in heart failure.31 A functional abnormality is also supported by studies evaluating the response to sympathetic nerve stimulation in vivo. Like postsynaptic -stimulation with epinephrine, stellate ganglion stimulation and eliciting presynaptic norepinephrine release using tyramine result in blunted rather than absent or hypersensitive contractile responses in hibernating myocardium.14 Teleologically, this could serve to uncouple local myocyte demand from increases in external workload during sympathetic activation to allow a regional balance between reduced oxygen delivery and reduced demand to be maintained during submaximal stress.
Limitations
To avoid potential effects of the stimulation on -receptor function, we did not evaluate contractile responses in the same animals. Individual correlation between cAMP production and in vivo functional responses was not possible. Nevertheless, our previous work in this well-characterized model demonstrates consistently attenuated contractile responses to epinephrine infusion in a number of studies.7eC9,14 More detailed in vivo studies assessing the functional consequences of changes in -receptor adenylyl cyclase coupling (eg, using higher doses and other agonists stimulating adenylyl cyclase activity) will require experiments where the effects of blunted -receptor function can be dissociated from ischemia. These challenging studies will necessitate experiments stimulating adenylyl cyclase through different pathways before and immediately after coronary revascularization of chronically instrumented animals. Finally, although the in vitro assays primarily reflect a cardiomyocyte population by volumetric fraction, there are small differences in connective tissue as well as membranes contributed by endothelium and vascular smooth muscle. The similarity of regional forskolin responses in hibernating and sham animals suggests that such tissue heterogeneity was not the cause of regional reductions in isoproterenol responses in hibernating myocardium.
Clinical Implications
Our data support the notion that the lack of contractile reserve during -adrenergic stimulation of viable dysfunctional myocardium can arise as the result of an intrinsic attenuation of -adrenergic signaling. This may be of considerable consequence in patients with ischemic cardiomyopathy where areas of normal, infarcted, and hibernating myocardium coexist within the same heart. The resultant heterogeneity in myocardial -adrenergic function could produce substantial inhomogeneity in myocardial repolarization, leading to a dynamic substrate factor for the development of ventricular tachycardia and fibrillation and sudden death during sympathetic activation. Indeed, patients with hibernating myocardium have a high short-term mortality32 that is paralleled by the high rate of sudden cardiac death caused by ventricular tachycardia and fibrillation in swine with hibernating myocardium.33
Finally, although further clinical correlation is required, our findings could explain the higher sensitivity of nuclear imaging as compared with assessing contractile reserve with -adrenergic stimulation to identify hibernating myocardium. This appears to be particularly problematic in the patient subgroup in whom resting flow is regionally reduced versus those in whom flow is normal.4,5 Thus, in patients with reduced resting flow, imaging viability with approaches that can ascertain the extent of infarction and fibrosis may be superior to imaging function during -adrenergic stimulation, which may be intrinsically reduced.
Acknowledgments
This work was supported by the Department of Veterans Affairs, the American Heart Association, the National Heart, Lung and Blood Institute (HL 55324 and HL 61610), the Albert and Elizabeth Rekate Fund, the Mae Stone Goode Trust, and the John R. Oishei Foundation. We appreciate the technical assistance of Anne Coe, Amy Johnson and Deana Gretka in these studies.
References
Bax JJ, Wijns W, Cornel JH, Visser FC, Boersma E, Fioretti PM. Accuracy of currently available techniques for prediction of functional recovery after revascularization in patients with left ventricular dysfunction due to chronic coronary artery disease: comparison of pooled data. J Am Coll Cardiol. 1997; 30: 1451eC1460.
Rahimtoola SH. Concept and evaluation of hibernating myocardium. Ann Rev Med. 1999; 50: 75eC86.
Heusch G, Schulz R, Rahimtoola SH. Myocardial hibernation: a delicate balance. Am J Physiol Heart Circ Physiol. 2005; 288: H984eCH999.
Melon PG, de Landsheere CM, Degueldre C, Peters JL, Kulbertus HE, Pierard LA. Relation between contractile reserve and positron emission tomographic patterns of perfusion and glucose utilization in chronic ischemic left ventricular dysfunction: implications for identification of myocardial viability. J Am Coll of Cardiol. 1997; 30: 1651eC1659.
Sawada S, Elsner G, Segar DS, O’Shaughnessy M, Khouri S, Foltz J, Bourdillon PD, Bates JR, Fineberg N, Ryan T, Hutchins GD, Feigenbaum H. Evaluation of patterns of perfusion and metabolism in dobutamine-responsive myocardium. J Am Coll Cardiol. 1997; 29: 55eC61.
Tawakol A, Skopicki HA, Abraham SA, Alpert NM, Fischman AJ, Picard MH, Gewirtz H. Evidence of reduced resting blood flow in viable myocardial regions with chronic asynergy. J Am Coll Cardiol. 2000; 36: 2146eC2153.
Fallavollita JA, Perry BJ, Canty JM Jr. 18F-2-deoxyglucose deposition and regional flow in pigs with chronically dysfunctional myocardium: evidence for transmural variations in chronic hibernating myocardium. Circulation. 1997; 95: 1900eC1909.
Malm BJ, Suzuki G, Canty JM Jr, Fallavollita JA. Variability of contractile reserve in hibernating myocardium: dependence on the method of stimulation. Cardiovasc Res. 2002; 56: 422eC433.
Fallavollita JA, Malm BJ, Canty JM Jr. Hibernating myocardium retains metabolic and contractile reserve despite regional reductions in flow, function, and oxygen consumption at rest. Circ Res. 2003; 92: 48eC55.
Shen YT, Kudej RK, Bishop SP, Vatner SF. Inotropic reserve and histological appearance of hibernating myocardium in conscious pigs with ameroid-induced coronary stenosis. Basic Res Cardiol. 1996; 91: 479eC485.
Sato S, Sato N, Kudej RK, Uechi M, Asai K, Shen YT, Ishikawa Y, Vatner SF, Vatner DE. -Adrenergic receptor signalling in stunned myocardium of conscious pigs. J Mol Cell Cardiol. 1997; 29: 1387eC1400.
Schulz R, Rose J, Martin C, Brodde O-E, Heusch G. Development of short-term myocardial hibernation: its limitation by the severity of ischemia and inotropic stimulation. Circulation. 1993; 88: 684eC695.
Shan K, Bick RJ, Poindexter BJ, Nagueh SF, Shimoni S, Verani MS, Keng F, Reardon MJ, Letsou GV, Howell JF, Zoghbi WA. Altered adrenergic receptor density in myocardial hibernation in humans. A possible mechanism of depressed myocardial function. Circulation. 2000; 102: 2599eC2606.
Ovchinnikov V, Suzuki G, Canty JM Jr, Fallavollita JA. Blunted functional responses to pre- and postjunctional sympathetic stimulation in hibernating myocardium. Am J Physiol Heart Circ Physiol. 2005; 289: H1719eCH1728.
Luisi AJ Jr, Fallavollita JA, Suzuki G, Canty JM Jr. Spatial inhomogeneity of sympathetic nerve function in hibernating myocardium. Circulation. 2002; 106: 779eC781.
Salomon Y, Londos C, Rodbell M. A highly sensitive adenylate cyclase assay. Anal Biochem. 1974; 58: 541eC548.
Fallavollita JA, Jacob SC, Young RF, Canty JM Jr. Regional alterations in SR Ca2+ATPase, phospholamban, and HSP-70 expression in chronic hibernating myocardium. Am J Physiol. 1999; 277: H1418eCH1428.
Hammond HK, White FC, Brunton LL, Longhurst JC. Association of decreased myocardial -receptors and chronotropic response to isoproterenol and exercise in pigs following chronic dynamic exercise. Circ Res. 1987; 60: 720eC726.
Boraso A, Ceconi C, Cargnoni A, Bernocchi P, Ferrari R, Ovize M. -Adrenergic receptors and intracellular signalling pathway in stunned and non-ischemic regions of pig myocardium. Basic Res Cardiol. 2001; 96: 388eC394.
Hammond HK, Roth DA, McKirnan MD, Ping P. Regional myocardial downregulation of the inhibitory guanosine triphosphate-binding protein (Gi2) and -adrenergic receptors in a porcine model of chronic episodic myocardial ischemia. J Clin Invest. 1993; 92: 2644eC2652.
Lefkowitz RJ. G protein-coupled receptor kinases: minireview. Cell. 1993; 74: 409eC412.
Freedman NJ, Liggett SB, Drachman DE, Pei G, Caron MG, Lefkowitz RJ. Phosphorylation and desensitization of the human beta 1-adrenergic receptor. Involvement of G protein-coupled receptor kinases and cAMP-dependent protein kinase. J Biol Chem. 1995; 270: 17953eC17961.
Benovic JL, Kuhn H, Weyand I, Codina J, Caron MG, Lefkowitz RJ. Functional desensitization of the isolated beta-adrenergic receptor by the beta-adrenergic receptor kinase: potential role of an analog of the retinal protein arrestin (48-kDa protein). Proc Natl Acad Sci U S A. 1987; 84: 8879eC8882.
Alousi AA, Jasper JR, Insel PA, Motulsky HJ. Stoichiometry of receptor-Gs-adenylate cyclase interactions. FASEB J. 1991; 5: 2300eC2303.
Ostrom RS, Gregorian C, Drenan RM, Xiang Y, Regan JW, Insel PA. Receptor number and caveolar colocalization determine receptor coupling efficiency to adenylyl cyclase. J Biol Chem. 2001; 276: 42063eC42069.
Port JD, Bristow MR. Altered beta-adrenergic receptor gene regulation and signaling in chronic heart failure. J Mol Cell Cardiol. 2001; 33: 887eC905.
Lim H, Fallavollita JA, Hard R, Kerr CW, Canty JM Jr. Profound apoptosis-mediated regional myocyte loss and compensatory hypertrophy in pigs with hibernating myocardium. Circulation. 1999; 100: 2380eC2386.
Nagata K, Communal C, Lim CC, Jain M, Suter TM, Eberli FR, Satoh N, Colucci WS, Apstein CS, Liao R. Altered -adrenergic signal transduction in nonfailing hypertrophied myocytes from Dahl salt-sensitive rats. Am J Physiol Heart Circ Physiol. 2000; 279: H2502eCH2508.
Luisi AJ Jr, Fallavollita JA, Suzuki G, deKemp R, Haka MS, Toorongian SA, Canty JM Jr. Regional 11C-hydroxyephedrine retention in hibernating myocardium: chronic inhomogeneity of sympathetic innervation in the absence of infarction. J Nucl Med. 2005; 46: 1368eC1374.
Vatner DE, Lavallee M, Amano J, Finizola A, Homcy CJ, Vatner SF. Mechanisms of supersensitivity to sympathomimetic amines in the chronically denervated heart of the conscious dog. Circ Res. 1985; 57: 55eC64.
Liang CS, Fan TH, Sullebarger JT, Sakamoto S. Decreased adrenergic neuronal uptake activity in experimental right heart failure. A chamber-specific contributor to beta-adrenoceptor downregulation. J Clin Invest. 1989; 84: 1267eC1275.
Di Carli MF, Asgarzadie F, Schelbert HR, Brunken RC, Rokhsar S, Maddahi J. Relation of myocardial perfusion at rest and during pharmacologic stress to the PET patterns of tissue viability in patients with severe left ventricular dysfunction. J Nucl Cardiol. 1998; 5: 558eC566.
Canty JM Jr, Suzuki G, Banas MD, Verheyen F, Borgers M, Fallavollita JA. Hibernating myocardium: chronically adapted to ischemia but vulnerable to sudden death. Circ Res. 2004; 94: 1142eC1149.(Vijay S. Iyer, John M. Ca)
the Departments of Medicine (V.S.I., J.M.C.) and Physiology & Biophysics (J.M.C.), University at Buffalo, NY.
Abstract
Contractile reserve during submaximal -adrenergic stimulation is attenuated in patients and swine with hibernating myocardium. We tested the hypothesis that this arises as a regional adaptive response in -adrenergic adenylyl cyclase coupling. Pigs (n=8) were studied 3 months after instrumentation with a left anterior descending artery (LAD) stenosis when flow (LAD, 0.7±0.2 versus 1.2±0.1 mL/min per gram in normal remote; P<0.05) and wall thickening (LAD, 5.5±3.2% versus 40.0±5.5% in remote; P<0.05) were reduced in the absence of infarction. Whereas basal cAMP production was normal (LAD, 87±18 versus 91±19 pmol/mg per minute; P=NS), responses to isoproterenol were blunted (LAD, 83±6 versus 146±25 pmol/mg per minute in remote; P<0.05). -receptor density and subtype were unchanged, but there was a reduction in the number of high-affinity binding sites (LAD, 40±4% versus 53±7% in normal remote; P<0.05). The Gi2/Gs ratio increased (LAD, 1.8±0.3 versus 0.99±0.3 in remote myocardium; P<0.05), although GppNHp-stimulated cAMP production was equivocally reduced. Forskolin responses were unchanged and similar to shams. These data indicate regional attenuation of -receptor adenylyl cyclase signaling in hibernating myocardium. This blunts the local contractile response to -adrenergic stimulation and may serve to protect against a myocardial supply/demand imbalance when external determinants of myocardial workload increase during sympathetic activation.
Key Words: -receptors hibernating myocardium chronic ischemic heart disease adenylyl cyclase
Introduction
Myocardial viability is an important determinant of prognosis in ischemic heart disease and is frequently identified by an increase in regional function in response to -adrenergic stimulation.1eC3 Unfortunately, a significant number of patients have limited contractile reserve when viability is present as assessed by nuclear techniques. The absence of contractile reserve appears to be particularly common in the subset of patients with hibernating myocardium where resting perfusion and function are both reduced, as compared with those with chronic stunning, where function is reduced and resting perfusion is normal.4eC6 We recently used a porcine model that progresses from stunned to chronic hibernating myocardium7 to demonstrate a similar attenuation of regional function during graded -adrenergic stimulation.8 Although the increase in flow was attenuated in the presence of a critical LAD stenosis, intrinsic adaptive responses in hibernating myocardium prevented the development of ischemia, as reflected by increased subendocardial function in the absence of lactate production during submaximal -adrenergic stimulation.9 These results suggest that hibernating myocardium operates at a lower point of the supply/demand relation than normal myocardium to limit an energetic imbalance during increases in external myocardial workload. The adaptations responsible for the attenuated contractile response to -adrenergic stimulation have not been established.
One potential adaptation that could explain the attenuated contractile reserve is a regional reduction in -adrenergic responsiveness arising from repetitive reversible ischemia. Although no previous studies have evaluated -receptor signaling in chronic hibernating myocardium, stunned myocardium has been reported to have increased -receptor density, little change in -receptoreCmediated cAMP production, and an accentuated contractile response.10,11 Although this is similar to the denervation hypersensitivity seen following transmural myocardial infarction, these findings have not been reproduced by others.12 Importantly, Shan et al actually demonstrated a reduction in -adrenergic surface density in needle biopsies from humans with hibernating myocardium.13 Such effects would be predicted to lead to an attenuated rather than accentuated contractile response to sympathetic stimulation and are consistent with in vivo observations in swine with hibernating myocardium.8,9,14
We, therefore, hypothesized that the attenuated metabolic and functional response to submaximal -adrenergic stimulation in pigs with hibernating myocardium arises from a regional downregulation in -adrenergic adenylyl cyclase coupling. To test this, we assessed in vitro -receptor density and affinity, stimulated cAMP production, and changes in regional G-protein expression in swine. The results demonstrate regional attenuation of -adrenergic signaling in hibernating myocardium that provides an intrinsic adaptation to limit the development of regional ischemia during increases in external workload.
Materials and Methods
Experimental procedures and protocols conformed to institutional guidelines for the care and use of animals in research. Hibernating myocardium was produced in pigs as previously described.7 Tissue for the present study was obtained from a series of animals used to assess in vivo myocardial norepinephrine uptake.15 Using sterile technique, juvenile pigs (n=8) were instrumented with a 1.5- to 2.0-mm fixed-diameter Delrin stenosis placed on the proximal left anterior descending artery (LAD). Three months after instrumentation, physiological studies were performed in the closed-chest sedated state (propofol 2 to 5 mg/kg per minute, IV supplemented with Telazol/xylazine).
We placed a 6F introducer into the brachial artery and inserted a 5F end-hole micromanometer (Millar Inc, Houston, Tex) into the left ventricle. Aortic pressure was monitored via the side port of the introducer. Blood flow measurements were determined at rest and during pharmacological vasodilation with adenosine (0.9 mg/kg per minute IV with phenylephrine coinfused to prevent hypotension) using fluorescent microspheres.8 Echocardiography was used to assess regional wall thickening in the hibernating LAD and normally perfused remote regions.8 End diastole was defined at the peak of the R wave and end systole was evaluated at minimum left ventricular (LV) chamber volume. The animals were allowed to recover and tissue sampling was performed at least 3 days after pharmacological stimulation.15 Tissue from the center of the hibernating region (adjacent to the LAD) was compared with normal remote LV myocardial samples taken adjacent to the posterior descending artery. Four additional sham animals were used to exclude changes in remote myocardium. Light microscopy and point counting were performed to quantify connective tissue.7
Membrane Preparation
Flash frozen (eC80°C) subendocardial samples were powdered in a stainless-steel mortar and pestle cooled with liquid nitrogen and homogenized using a polytron (setting, 4 to 5) in a buffer containing 50 mmol/L Tris (pH 7.4), 20% sucrose, 2 mmol/L EGTA, 10 e/mL Leupeptin, 10 e/mL pepstatin, 10 e/mL aprotinin, and 5 e/mL phenylmethylsulfonyl fluoride. After homogenates were centrifuged (10 minutes; 2500g), we centrifuged the supernatant (20 minutes; 48 000g). Pellets were resuspended in 50 mmol/L Tris or 30 mmol/L HEPES (pH 7.4) and frozen at eC80°C for subsequent use in radioligand-binding experiments, adenylyl cyclase assays, and Western blot analysis. Membrane protein concentrations were determined in triplicate and yields were similar in LAD and remote regions (9.9±0.5 mg/g versus 10±0.5 mg/g; P=NS).
Adenylyl Cyclase Activity
Adenylyl cyclase activity was determined using a modification of the method by Salomon et al.16 Assays were performed in 100 e蘈 of solution containing 15 mmol/L HEPES (pH 7.4), 5 mmol/L MgCl2, 0.4 mmol/L ATP, 5 mmol/L creatine phosphate, 0.5 mmol/L EGTA, 50 mmol/L NaCl, 1 mmol/L cAMP, 60 U/mL creatine phosphokinase, 1 mmol/L 3-iso-butyl-2-methylxanthine, and 2 e藽i [32P]ATP. Reactions were initiated by adding 100 e of membrane protein and incubated at 37°C for 20 minutes. The reaction was terminated by adding 100 e蘈 of stopping solution (2% sodium dodecyl sulfate, 40 mmol/L ATP, and 1.4 mmol/L cAMP). We added [3H] (5000 counts per minute) to each reaction for estimation of column recovery. The [32P]cAMP produced was separated from [32P]ATP by sequential Dowex and alumina chromatography. Column recovery was 85% to 95%. Adenylyl cyclase activity was determined under basal conditions and after stimulation with isoproterenol (10eC4 mol/L in the presence of 0.1 mmol/L GTP), forskolin (10eC4 mol/L), and GppNHp (10eC4 mol/L). In preliminary experiments, we performed dose-response curves with each of these agents to determine the concentration eliciting maximal adenylyl cyclase cAMP production for use in subsequent studies. Data represent total cAMP production. Baseline cAMP production before stimulation was not different from the basal state (P>0.05, ANOVA).
Western Blotting
Regional expression of G proteins (25 e/lane) and G-protein receptor kinase (GRK) (40 e/lane) was determined on membrane preparations as previously described.17 Paired experimental and normal samples were run simultaneously to minimize variability. Equal loading and transfer were confirmed in preliminary experiments (Coomassie blue and Ponceau S staining). Nitrocellulose membranes were incubated with antibodies (Santa Cruz Biotechnology Inc) to G proteins (Gs [SC-823], 1:1000; Gi2 [SC-391], 1:1000 dilution) and GRK-2 and GRK-5 (GRK-2 [SC-562] and GRK-5 [SC-565]; each at 1:500 dilution) overnight at 4°C. They were subsequently incubated for 1 hour with 0.1% TBS containing horseradish peroxidaseeCconjugated anti-rabbit IgG at 1:10 000 dilutions, visualized with the Supersignal West Peco chemiluminescent substrate (Pierce, Ill), and quantified with a laser densitometer (Bio-Rad Inc).
Radioligand Binding Experiments
-Adrenergic receptors were quantified using 125I-iodocyanopindolol (5 to 450 pmol/L) in saturation isotherm experiments conducted in membrane preparations as previously described.18 In brief, 100 e of membrane protein prepared as described above was suspended in binding buffer containing 250 mmol/L HEPES, 20 mmol/L MgCl2, and 0.2% BSA. The membranes were incubated with increasing concentrations of 125I-iodocyanopindolol for 1 hour at 37°C in a water bath. Nonspecific binding was defined as binding in the presence of 10 eol/L propranolol. All reactions were performed in triplicate and specific binding was >80%. The reactions were terminated by rapid filtration using a Brandel M-24R cell harvester (Gaithersburg, Md) through Whatman GF/C filters followed by 4 washes with 4 mL of wash buffer (20 mmol/L Tris-HCl [pH 7.4], 2 mmol/L MgCl2). The filters were transferred to polystyrene tubes and counted in a gamma counter for 1 minute. The antagonist binding data were analyzed using the Graph Pad Prism program. The proportion of receptors in high-affinity and low-affinity states were determined in competition-binding experiments by incubating 100 pmol/L 125I-iodocyanopindolol with sixteen increasing concentrations of isoproterenol (10eC11 to 10eC4 mol/L) in the absence of guanine nucleotides. The binding assays were performed in the buffer described above with 3-hour incubation at 30°C. All reactions were performed in duplicate, with nonspecific binding assessed in the presence of 0.1 mmol/L isoproterenol. The displacement curves were fit to 1-site or 2-site binding models using the Graph Pad Prism 4 curve-fitting program. A 1-way ANOVA compared 2- and 1-site fits. Finally, proportions of 1 and 2 receptors were determined in competition-binding experiments using 16 different concentrations of CGP20712A (10eC11 to 10eC4 mol/L), a selective 1 antagonist.
Statistical Analysis
Data are reported as the mean±SEM. After an ANOVA, post hoc analysis was performed with paired 2-tailed t tests. Unpaired t tests were used for sham comparisons. A value of P<0.05 was significant.
Results
All pigs were in good health at the time of study and 2eC3-5-triphenyl tetrazolium chloride staining showed no evidence of acute or healed infarction. Angiography demonstrated total occlusion of the LAD, which filled by collaterals arising from both left and right coronary arteries. There was anterior hypokinesis consistent with viable dysfunctional myocardium. Although point counting demonstrated an increase in interstitial connective tissue typical of hibernating myocardium (7.3±1.4% versus 3.6±0.5% in remote regions; P<0.05), >90% of both regions was comprised of myocytes.
Under resting conditions, heart rate averaged 94±7 bpm, LV systolic pressure averaged 147±7 mm Hg, and LV end-diastolic pressure averaged 23±2 mm Hg. Measurements of resting flow and wall thickening confirmed the presence of hibernating myocardium. In the subendocardium (Figure 1A), flow was reduced at rest (LAD, 0.75±0.14 versus 1.19±0.14 mL/min per gram in normal remote myocardium; P<0.05). Like the hypokinesis demonstrated on ventriculography, LAD wall thickening was also significantly reduced (Figure 1B; LAD, 5.5±3.2% versus 40.0±5.5% in remote; P<0.05). Following adenosine, subendocardial flow in the hibernating region was critically impaired (0.7±0.2 mL/min per gram) and unable to increase above resting values, whereas flow in the remote region increased 4 times (4.7±0.8 mL/min per gram; P<0.05, LAD versus remote). Thus, subendocardial samples had physiological features of hibernating myocardium in the absence of infarction.
Adenylyl Cyclase Activity
Figure 2 summarizes results of the adenylyl cyclase activity assays. Total cAMP accumulation was similar under basal conditions (LAD, 87±18 versus 91±19 pmol/mg per minute in remote). Stimulation with isoproterenol (10eC4 mol/L) resulted in an attenuated cAMP accumulation in hibernating myocardium (LAD, 83±6 versus 146±25 pmol/mg per minute in remote; P<0.05). Stimulation of adenylyl cyclase with GppNHp (10eC4 mol/L) was also attenuated in hibernating myocardium, but the difference was of borderline significance (LAD, 202±37 versus 277±64 pmol/mg per minute in remote; P=0.11). Forskolin-stimulated responses (10eC4 mol/L) were not different (LAD, 276±39 versus 299±43 pmol/mg per minute in remote; P=NS). Thus, there was a regional attenuation of -mediated stimulation of adenylyl cyclase and normal stimulation with forskolin in chronic hibernating myocardium.
In sham instrumented pigs, basal (LAD, 103±19 versus 97±22 pmol/mg per minute in remote; P=NS), isoproterenol-stimulated (LAD, 156±14 versus 152±18 pmol/mg per minute in remote; P=NS), and forskolin-stimulated (LAD, 348±31 versus 335±28 pmol/mg per minute in remote; P=NS) cAMP were similar. Remote myocardial values in shams were not significantly different from remote values in animals with hibernating myocardium.
G-Protein and GRK Immunoblotting
Analysis of G protein levels (Figure 3) revealed an increase in Gi2 levels in hibernating myocardium (LAD, 11.2±2.2 versus 7.2±1.2 densitometric units in remote; P<0.05). There was a trend toward reduced membrane-associated levels of Gs, which did not reach statistical significance (LAD, 7.7±1.2 versus 12.2±3.3 densitometric units in remote; P=0.15). There were no regional differences in Gi2 or Gs proteins in sham animals. Thus, the Gi2/Gs ratio in hibernating regions was significantly increased (LAD, 1.84±0.3 versus 0.9±0.3 in remote myocardium; P<0.005). GRK-2 protein levels were similar in remote and hibernating tissue, whereas GRK-5 protein showed a trend toward increasing in hibernating myocardium (LAD, 7.8±1.2 versus 6.8±1.1 densitometric units in remote; P=0.15).
Radioligand Binding
The Table summarizes results of radioligand-binding experiments. Scatchard analyses r2 values were 0.96±0.01. There was no difference in total -receptoreCbinding sites using 125I-iodocyanopindolol in hibernating versus remote myocardium. Displacement experiments using 125I-iodocyanopindolol to assess changes in high- and low-affinity receptors (Figure 4) consistently fit best to a 2-site model (ANOVA, P<0.05) and were shifted to the right in hibernating myocardium. This reflected a reduction in the number of high-affinity binding sites in hibernating myocardium (40±4% versus 53±7%; P<0.05). Competition-binding experiments done in the presence of increasing concentrations of CGP20712A (Figure 5), a selective 1 antagonist, did not reveal any differences in the proportion of 1 and 2 receptor subtypes (Table).
Discussion
There are several new and important findings from our study. First, there is a regional attenuation of -adrenergiceCmediated cAMP production in subendocardial samples from hibernating myocardium that occurs in the absence of infarction. This was not attributable to alterations in adenylyl cyclase activity because direct stimulation with forskolin was not affected. Second, whereas there was no alteration in total -receptor density in vitro, competition-binding curves demonstrated a reduction in -receptor affinity. This was associated with a regional reduction in the number of high-affinity binding sites and increase in Gi2 proteins. Collectively, our results support the notion that an adaptive downregulation of -adrenergic responsiveness contributes to attenuating increases in regional metabolic demand during increases in external workload from sympathetic activation.
Effects of Reversible Ischemia on -Adrenergic Signaling
The effects of ischemia on -receptor signaling have been studied in a variety of models of reversible transmural ischemia leading to stunned myocardium. Although the specific alterations depend on the model used, as well as the duration of ischemia, most of the available data have demonstrated no change or evidence of accentuated -receptor adenylyl cyclase coupling. For example, Sato et al found wall thickening after isoproterenol increased more in stunned myocardium than in control, a finding potentially consistent with -adrenergic hypersensitivity following ischemia.11 Whereas this was associated with a small increase in -receptor density, isoproterenol-stimulated cAMP production was unchanged. In chronically stunned myocardium, in vivo -adrenergic stimulation normalized regional function, although effects on -adrenergic adenylyl cyclase production were not reported.10 In a model using multiple brief episodes of ischemia, Boraso et al found no changes in cAMP, -receptor density, affinity, or G-protein levels.19 Likewise, prolonged moderate ischemia or short-term hibernation in swine was associated with contractile reserve to dobutamine and unchanged -receptor levels, with no evidence of denervation hypersensitivity.12 Thus, following reversible ischemia, the functional response to transient -adrenergic stimulation is either normal or accentuated and biochemical evidence of alterations in cellular -receptor signaling is limited.
Hammond et al studied -receptor signaling in pigs subjected to repetitive ischemia from an ameroid constrictor placed around the left circumflex artery.20 Although demand-induced ischemia was produced in their model, the physiological significance of the stenosis was not sufficient to cause chronic reductions in resting flow or function. Thus, these investigators were able to evaluate the effects of repetitive ischemia on -adrenergic adenylyl cyclase coupling independently of the progression to stunned or hibernating myocardium. Like the present study, they reported a regional reduction in -receptor affinity with a shift to a low-affinity state in dysfunctional myocardium. Although these changes were accompanied by a reduction in -receptor number, there were no effects on stimulated adenylyl cyclase activity. These paradoxical findings were explained by a reciprocal reduction in Gi2 and an increase in Gs compensating for the alteration in -receptor number and affinity.
Our results in hibernating myocardium differ from those reported in myocardium subjected to reversible ischemia with or without stunning in several respects. We found that cAMP production in response to isoproterenol stimulation was regionally reduced in chronic hibernating myocardium. This supports previous in vivo functional observations in this model demonstrating an attenuated contractile response to -adrenergic stimulation with epinephrine when assessed using regional wall motion by ventriculography,7 regional wall-thickening measurements from echocardiography,8 and subendocardial segment length shortening.9,14 In each circumstance, -adrenergic stimulation improved but did not normalize myocardial function in hibernating myocardium. Radioligand-binding data in the presence of 125I-cyanopindolol demonstrated that Bmax (total receptor binding sites) was similar in remote and hibernating regions, indicating that total -adrenergic receptor density was normal. Competition binding revealed a loss of high-affinity receptor binding sites in hibernating myocardium, which at least partly explained the reduced ability of -adrenergic stimulation to increase cAMP in response to isoproterenol. The mechanism for the loss of high-affinity sites is most likely an uncoupling of the receptor from the G-protein. While speculative, phosphorylation of the receptor by protein kinases, including G-protein coupled receptor kinases, could cause uncoupling of the receptoreCG-protein complex.21eC23 Although the increase in GRK-5 levels was not significant, and GRK-2 protein levels were unchanged, it is plausible that their activities are significantly enhanced in hibernating myocardium and will require further studies.
Role of Altered G-Protein Expression on Adenylyl Cyclase in Hibernating Myocardium
The differences in -adrenergic signaling caused by chronic ischemia and hibernating myocardium lead to opposing effects of these states on G-protein expression. We found reciprocal alterations in the levels of G proteins in hibernating myocardium with the inhibitory subunit Gi2 increasing and the stimulatory subunit Gs decreasing. These are the exact opposite of the changes reported in myocardium with normal resting function subjected to chronic repetitive ischemia by Hammond et al.20 Coupled with the differences in -adrenergiceCmediated cAMP production, the results support the notion that hibernating myocardium reflects a fundamentally different phase of the chronic adaptive response to repetitive ischemia.
The functional importance of alterations in G-protein levels in hibernating myocardium is unclear because relative protein changes were greater than the reduction in GppNHp-stimulated adenylyl cyclase activity. This could reflect the possibility that G-protein changes have no functional impact because of the molar excess of G-proteins as compared with receptors and adenylyl cyclase molecules.24 Alternatively, it could reflect differential activation of the individual G-proteins or compartmentalization of a pool of G-proteins that, along with receptors and adenylyl cyclase molecules, colocalize in microdomains of the cell membrane lined by caveolar proteins.25 Thus, the stoichiometric excess of G-proteins in the whole cell might not represent the pool of G-proteins available to the receptors in caveolar microdomains. Finally, it is conceivable that there is an effect on the dose-response relation to GppNHp that could be brought out at submaximal concentrations, which we did not study. Further studies will be required to determine the functional impact of chronic alterations in G-protein expression because these changes may impact on other adaptive responses in hibernating myocardium.
Relation of Postsynaptic Reductions in -Adrenergic Receptor Signaling to Cellular Hypertrophy and Reductions in Presynaptic Norepinephrine Uptake
Although regional, our findings in hibernating myocardium are reminiscent of those reported in models of heart failure, where there are global reductions in -receptor signaling in the presence of systemic neurohormonal activation and reduced presynaptic norepinephrine uptake.26 In some heart failure models, these changes are accompanied by global reductions in the expression of the sarcoplasmic reticulum calcium-uptake proteins, phospholamban, and the sarcoplasmic reticulum Ca2+ATPase, which is seen regionally in the absence of neurohormonal activation and heart failure in hibernating myocardium.17 Whereas global cellular hypertrophy occurs in the failing heart, hibernating myocardium is associated with regional hypertrophy secondary to low but chronic rates of apoptosis.27 A number of laboratories have demonstrated altered -adrenergic adenylyl cyclase coupling in models of cellular hypertrophy that are not associated with decompensation or neurohormonal activation.28 Thus, it is conceivable that the alterations in -adrenergic affinity and attenuated cAMP production reflect a common response of the hypertrophied cardiac myocyte.
Although the presence of normal -adrenergic signaling in remote regions from the same heart excludes systemic neurohormonal activation as a mechanism in the present study, compartmental increases in norepinephrine are an alternative mechanism leading to the regional downregulation in -receptor signaling. We have previously demonstrated that regional norepinephrine uptake is moderately reduced in hibernating myocardium, as assessed by the uptake of 125IeCmeta-iodo-benzylguanidine ex vivo15 and imaging 11C-hydroxyephedrine uptake using positron emission tomography in vivo.29 These regional changes may reflect a loss of sympathetic nerves or a functional impairment in norepinephrine uptake. If reduced norepinephrine uptake was indicative of partial sympathetic denervation, it would be predicted to result in accentuated functional responses and increased -adrenergiceCmediated cAMP production. Although such effects have been seen several weeks after surgical sympathetic denervation in animals,30 the blunted cAMP production and functional responses during -adrenergic stimulation argue against this mechanism in hibernating myocardium.
Our results support a fundamentally different postsynaptic response than that seen in the denervated heart. The most likely explanation is that alterations in 125IeCmeta-iodo-benzylguanidine and 11C-hydroxyephedrine reflect chronic functional abnormalities in the presynaptic norepinephrine uptake mechanism rather than denervation. In this circumstance, locally increased norepinephrine overflow during sympathetic activation would be predicted to lead to a regional downregulation in -adrenergic receptor function rather than -adrenergic hypersensitivity. A similar mechanism has been hypothesized to contribute to chamber-specific -receptor downregulation in heart failure.31 A functional abnormality is also supported by studies evaluating the response to sympathetic nerve stimulation in vivo. Like postsynaptic -stimulation with epinephrine, stellate ganglion stimulation and eliciting presynaptic norepinephrine release using tyramine result in blunted rather than absent or hypersensitive contractile responses in hibernating myocardium.14 Teleologically, this could serve to uncouple local myocyte demand from increases in external workload during sympathetic activation to allow a regional balance between reduced oxygen delivery and reduced demand to be maintained during submaximal stress.
Limitations
To avoid potential effects of the stimulation on -receptor function, we did not evaluate contractile responses in the same animals. Individual correlation between cAMP production and in vivo functional responses was not possible. Nevertheless, our previous work in this well-characterized model demonstrates consistently attenuated contractile responses to epinephrine infusion in a number of studies.7eC9,14 More detailed in vivo studies assessing the functional consequences of changes in -receptor adenylyl cyclase coupling (eg, using higher doses and other agonists stimulating adenylyl cyclase activity) will require experiments where the effects of blunted -receptor function can be dissociated from ischemia. These challenging studies will necessitate experiments stimulating adenylyl cyclase through different pathways before and immediately after coronary revascularization of chronically instrumented animals. Finally, although the in vitro assays primarily reflect a cardiomyocyte population by volumetric fraction, there are small differences in connective tissue as well as membranes contributed by endothelium and vascular smooth muscle. The similarity of regional forskolin responses in hibernating and sham animals suggests that such tissue heterogeneity was not the cause of regional reductions in isoproterenol responses in hibernating myocardium.
Clinical Implications
Our data support the notion that the lack of contractile reserve during -adrenergic stimulation of viable dysfunctional myocardium can arise as the result of an intrinsic attenuation of -adrenergic signaling. This may be of considerable consequence in patients with ischemic cardiomyopathy where areas of normal, infarcted, and hibernating myocardium coexist within the same heart. The resultant heterogeneity in myocardial -adrenergic function could produce substantial inhomogeneity in myocardial repolarization, leading to a dynamic substrate factor for the development of ventricular tachycardia and fibrillation and sudden death during sympathetic activation. Indeed, patients with hibernating myocardium have a high short-term mortality32 that is paralleled by the high rate of sudden cardiac death caused by ventricular tachycardia and fibrillation in swine with hibernating myocardium.33
Finally, although further clinical correlation is required, our findings could explain the higher sensitivity of nuclear imaging as compared with assessing contractile reserve with -adrenergic stimulation to identify hibernating myocardium. This appears to be particularly problematic in the patient subgroup in whom resting flow is regionally reduced versus those in whom flow is normal.4,5 Thus, in patients with reduced resting flow, imaging viability with approaches that can ascertain the extent of infarction and fibrosis may be superior to imaging function during -adrenergic stimulation, which may be intrinsically reduced.
Acknowledgments
This work was supported by the Department of Veterans Affairs, the American Heart Association, the National Heart, Lung and Blood Institute (HL 55324 and HL 61610), the Albert and Elizabeth Rekate Fund, the Mae Stone Goode Trust, and the John R. Oishei Foundation. We appreciate the technical assistance of Anne Coe, Amy Johnson and Deana Gretka in these studies.
References
Bax JJ, Wijns W, Cornel JH, Visser FC, Boersma E, Fioretti PM. Accuracy of currently available techniques for prediction of functional recovery after revascularization in patients with left ventricular dysfunction due to chronic coronary artery disease: comparison of pooled data. J Am Coll Cardiol. 1997; 30: 1451eC1460.
Rahimtoola SH. Concept and evaluation of hibernating myocardium. Ann Rev Med. 1999; 50: 75eC86.
Heusch G, Schulz R, Rahimtoola SH. Myocardial hibernation: a delicate balance. Am J Physiol Heart Circ Physiol. 2005; 288: H984eCH999.
Melon PG, de Landsheere CM, Degueldre C, Peters JL, Kulbertus HE, Pierard LA. Relation between contractile reserve and positron emission tomographic patterns of perfusion and glucose utilization in chronic ischemic left ventricular dysfunction: implications for identification of myocardial viability. J Am Coll of Cardiol. 1997; 30: 1651eC1659.
Sawada S, Elsner G, Segar DS, O’Shaughnessy M, Khouri S, Foltz J, Bourdillon PD, Bates JR, Fineberg N, Ryan T, Hutchins GD, Feigenbaum H. Evaluation of patterns of perfusion and metabolism in dobutamine-responsive myocardium. J Am Coll Cardiol. 1997; 29: 55eC61.
Tawakol A, Skopicki HA, Abraham SA, Alpert NM, Fischman AJ, Picard MH, Gewirtz H. Evidence of reduced resting blood flow in viable myocardial regions with chronic asynergy. J Am Coll Cardiol. 2000; 36: 2146eC2153.
Fallavollita JA, Perry BJ, Canty JM Jr. 18F-2-deoxyglucose deposition and regional flow in pigs with chronically dysfunctional myocardium: evidence for transmural variations in chronic hibernating myocardium. Circulation. 1997; 95: 1900eC1909.
Malm BJ, Suzuki G, Canty JM Jr, Fallavollita JA. Variability of contractile reserve in hibernating myocardium: dependence on the method of stimulation. Cardiovasc Res. 2002; 56: 422eC433.
Fallavollita JA, Malm BJ, Canty JM Jr. Hibernating myocardium retains metabolic and contractile reserve despite regional reductions in flow, function, and oxygen consumption at rest. Circ Res. 2003; 92: 48eC55.
Shen YT, Kudej RK, Bishop SP, Vatner SF. Inotropic reserve and histological appearance of hibernating myocardium in conscious pigs with ameroid-induced coronary stenosis. Basic Res Cardiol. 1996; 91: 479eC485.
Sato S, Sato N, Kudej RK, Uechi M, Asai K, Shen YT, Ishikawa Y, Vatner SF, Vatner DE. -Adrenergic receptor signalling in stunned myocardium of conscious pigs. J Mol Cell Cardiol. 1997; 29: 1387eC1400.
Schulz R, Rose J, Martin C, Brodde O-E, Heusch G. Development of short-term myocardial hibernation: its limitation by the severity of ischemia and inotropic stimulation. Circulation. 1993; 88: 684eC695.
Shan K, Bick RJ, Poindexter BJ, Nagueh SF, Shimoni S, Verani MS, Keng F, Reardon MJ, Letsou GV, Howell JF, Zoghbi WA. Altered adrenergic receptor density in myocardial hibernation in humans. A possible mechanism of depressed myocardial function. Circulation. 2000; 102: 2599eC2606.
Ovchinnikov V, Suzuki G, Canty JM Jr, Fallavollita JA. Blunted functional responses to pre- and postjunctional sympathetic stimulation in hibernating myocardium. Am J Physiol Heart Circ Physiol. 2005; 289: H1719eCH1728.
Luisi AJ Jr, Fallavollita JA, Suzuki G, Canty JM Jr. Spatial inhomogeneity of sympathetic nerve function in hibernating myocardium. Circulation. 2002; 106: 779eC781.
Salomon Y, Londos C, Rodbell M. A highly sensitive adenylate cyclase assay. Anal Biochem. 1974; 58: 541eC548.
Fallavollita JA, Jacob SC, Young RF, Canty JM Jr. Regional alterations in SR Ca2+ATPase, phospholamban, and HSP-70 expression in chronic hibernating myocardium. Am J Physiol. 1999; 277: H1418eCH1428.
Hammond HK, White FC, Brunton LL, Longhurst JC. Association of decreased myocardial -receptors and chronotropic response to isoproterenol and exercise in pigs following chronic dynamic exercise. Circ Res. 1987; 60: 720eC726.
Boraso A, Ceconi C, Cargnoni A, Bernocchi P, Ferrari R, Ovize M. -Adrenergic receptors and intracellular signalling pathway in stunned and non-ischemic regions of pig myocardium. Basic Res Cardiol. 2001; 96: 388eC394.
Hammond HK, Roth DA, McKirnan MD, Ping P. Regional myocardial downregulation of the inhibitory guanosine triphosphate-binding protein (Gi2) and -adrenergic receptors in a porcine model of chronic episodic myocardial ischemia. J Clin Invest. 1993; 92: 2644eC2652.
Lefkowitz RJ. G protein-coupled receptor kinases: minireview. Cell. 1993; 74: 409eC412.
Freedman NJ, Liggett SB, Drachman DE, Pei G, Caron MG, Lefkowitz RJ. Phosphorylation and desensitization of the human beta 1-adrenergic receptor. Involvement of G protein-coupled receptor kinases and cAMP-dependent protein kinase. J Biol Chem. 1995; 270: 17953eC17961.
Benovic JL, Kuhn H, Weyand I, Codina J, Caron MG, Lefkowitz RJ. Functional desensitization of the isolated beta-adrenergic receptor by the beta-adrenergic receptor kinase: potential role of an analog of the retinal protein arrestin (48-kDa protein). Proc Natl Acad Sci U S A. 1987; 84: 8879eC8882.
Alousi AA, Jasper JR, Insel PA, Motulsky HJ. Stoichiometry of receptor-Gs-adenylate cyclase interactions. FASEB J. 1991; 5: 2300eC2303.
Ostrom RS, Gregorian C, Drenan RM, Xiang Y, Regan JW, Insel PA. Receptor number and caveolar colocalization determine receptor coupling efficiency to adenylyl cyclase. J Biol Chem. 2001; 276: 42063eC42069.
Port JD, Bristow MR. Altered beta-adrenergic receptor gene regulation and signaling in chronic heart failure. J Mol Cell Cardiol. 2001; 33: 887eC905.
Lim H, Fallavollita JA, Hard R, Kerr CW, Canty JM Jr. Profound apoptosis-mediated regional myocyte loss and compensatory hypertrophy in pigs with hibernating myocardium. Circulation. 1999; 100: 2380eC2386.
Nagata K, Communal C, Lim CC, Jain M, Suter TM, Eberli FR, Satoh N, Colucci WS, Apstein CS, Liao R. Altered -adrenergic signal transduction in nonfailing hypertrophied myocytes from Dahl salt-sensitive rats. Am J Physiol Heart Circ Physiol. 2000; 279: H2502eCH2508.
Luisi AJ Jr, Fallavollita JA, Suzuki G, deKemp R, Haka MS, Toorongian SA, Canty JM Jr. Regional 11C-hydroxyephedrine retention in hibernating myocardium: chronic inhomogeneity of sympathetic innervation in the absence of infarction. J Nucl Med. 2005; 46: 1368eC1374.
Vatner DE, Lavallee M, Amano J, Finizola A, Homcy CJ, Vatner SF. Mechanisms of supersensitivity to sympathomimetic amines in the chronically denervated heart of the conscious dog. Circ Res. 1985; 57: 55eC64.
Liang CS, Fan TH, Sullebarger JT, Sakamoto S. Decreased adrenergic neuronal uptake activity in experimental right heart failure. A chamber-specific contributor to beta-adrenoceptor downregulation. J Clin Invest. 1989; 84: 1267eC1275.
Di Carli MF, Asgarzadie F, Schelbert HR, Brunken RC, Rokhsar S, Maddahi J. Relation of myocardial perfusion at rest and during pharmacologic stress to the PET patterns of tissue viability in patients with severe left ventricular dysfunction. J Nucl Cardiol. 1998; 5: 558eC566.
Canty JM Jr, Suzuki G, Banas MD, Verheyen F, Borgers M, Fallavollita JA. Hibernating myocardium: chronically adapted to ischemia but vulnerable to sudden death. Circ Res. 2004; 94: 1142eC1149.(Vijay S. Iyer, John M. Ca)