doi: 10.1161/hc4201.097188
(Circulation. 2001;104:2069.)
© 2001 American Heart Association, Inc.
Basic Science Reports |
Ventricular Dysfunction After Cardioplegic Arrest Is Improved After Myocardial Gene Transfer of a ß-Adrenergic Receptor Kinase Inhibitor
From the Departments of Surgery (H.T.T., A.D.E., K.F.S., W.J.K.), Pharmacology and Cancer Biology (W.J.K.), and Medicine (K.W.) and the Howard Hughes Medical Institutes (K.W.), Duke University Medical Center, Durham, NC.
Correspondence to Walter J. Koch, PhD, Department of Surgery, Box 2606, MSRB Room 471, Duke University Medical Center, Durham, NC 27710. E-mail koch0002{at}mc.duke.edu
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Abstract |
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Background— Acute cardiac contractile dysfunction is common after cardiopulmonary bypass (CPB). A potential molecular mechanism is enhanced ß-adrenergic receptor kinase (ßARK1) activity, because ß-adrenergic receptor (ßAR) signaling is altered in cardiomyocytes after cardioplegia. Therefore, we examined whether adenovirus-mediated intracoronary delivery of a ßARK1 inhibitor (Adv-ßARKct) could prevent post-CPB dysfunction.
Methods and Results— Rabbits were randomized to receive 5x1011 total viral particles of Adv-ßARKct or PBS. After 5 days, hearts were arrested with University of Wisconsin solution, excised, and stored at 4°C for 15 minutes or 4 hours before reperfusion on a Langendorff apparatus. Left ventricular (LV) function measured by end-diastolic pressure response to preload augmentation, contractility (LV dP/dtmax), and relaxation (LV dP/dtmin) was assessed by use of increasing doses of isoproterenol and compared with a control group of nonarrested hearts acutely perfused on the Langendorff apparatus. In the PBS-treated hearts, LV function decreased in a temporal manner and was significantly impaired compared with control hearts after 4 hours of cardioplegic arrest. LV function in Adv-ßARKct-treated hearts, however, was significantly enhanced compared with PBS treatment and was similar to control nonarrested hearts even after 4 hours of cardioplegia. Biochemically, several aspects of ßAR signaling were dysfunctional in PBS-treated hearts, whereas they were normalized in ßARKct-overexpressing hearts.
Conclusions— Myocardial gene transfer of Adv-ßARKct stabilizes ßAR signaling and prevents LV dysfunction induced by prolonged cardioplegic arrest. Thus, ßARK1 inhibition may represent a novel target in limiting depressed ventricular function after CPB.
Key Words: ischemia • reperfusion • cardiopulmonary bypass • gene therapy • signal transduction
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Introduction |
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Cold cardioplegic cardiac arrest, either during cardiopulmonary bypass (CPB) procedures or during harvest and transport of a donor heart for transplantation, induces a degree of cardiac dysfunction by the time the heart is reperfused. Left ventricular (LV) compliance as well as LV contractility and relaxation are often impaired, which necessitates the use of inotropic agents for postoperative hemodynamic support.1 At a molecular level, the myocardial ß-adrenergic receptor (ßAR) signaling system is crippled during the cold ischemic period and is not immediately restored by warm blood reperfusion.2–6 Importantly, cardiac ßARs are the most powerful means to support myocardial contractile function, and thus, the inotropic reserve of postsurgery hearts is lost.
Both ß1- and ß2-ARs are desensitized during CPB, which might be a direct result of a large local release of catecholamines.2–4 In addition, although total ßAR density does not change in human atrial samples taken before, during, or after CPB, ß-agonist–stimulated adenylyl cyclase activity is decreased, suggesting an important role for ßAR uncoupling.2–6 The ß-adrenergic receptor kinase (ßARK1 or GRK2), a member of the G protein–coupled receptor kinase family (GRK), is responsible for phosphorylating and uncoupling agonist-occupied ßARs.7 Interestingly, ßARK1 expression and activity are increased in different forms of heart disease, including heart failure8 and myocardial ischemia.9 Consistent with this is the finding that cardiac ßARK1 expression can also be increased after acute catecholamine exposure.10 Thus, ßARK1 may be the key molecule in initiating ßAR uncoupling early after cardioplegic arrest.
ßARK1 activity has been shown to be elevated in models of ischemia-reperfusion,9,11 and we recently demonstrated that myocardial recovery after ischemia-reperfusion injury is significantly impaired in transgenic mice overexpressing ßARK1.12 Although cold cardioplegic solutions are supposed to protect the heart, some similarities may exist between ischemia-reperfusion and cardioplegic cardiac arrest followed by warm blood reperfusion. Accordingly, we hypothesized that inhibition of ßARK1 may represent a new strategy to prevent myocardial dysfunction after reperfusion of cardioplegia-arrested hearts. We previously developed a peptide that inhibits ßARK1 activity (ßARKct).13–15 The ßARKct is a 194-amino-acid peptide corresponding to the carboxyl terminus end of ßARK1, and it includes the sequence responsible for binding to the ß
-subunit of activated heterotrimeric G proteins (Gß), a process required for ßARK1 activity.13–15 In the present study, we demonstrate that intracoronary adenovirus-mediated gene transfer of the ßARKct (Adv-ßARKct) before cardioplegic arrest prevents LV dysfunction by the time the heart is reperfused.
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Methods |
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Adenoviral Constructs
The adenoviral backbone for Adv-ßARKct is a second-generation replication-deficient serotype 2 adenovirus with deletions of the E1 and E4 (except for ORF6), as previously described.16,17 Aliquots of 5x1011 total viral particles (TVP) were thawed and mixed in PBS for a final volume of 2 mL immediately before intracoronary delivery.
In Vivo Intracoronary Gene Delivery
Adult male New Zealand White rabbits (3 kg) were operated on as previously described.17,18 Procedures were humanely performed in accordance with the regulations adopted by the National Institutes of Health and approved by the Animal Care and Use Committee of Duke University. To control for ßARKct expression, a group of animals randomly received 2 mL of PBS by use of the same delivery technique.
Isolated Heart Preparation and LV Function Assessment
Five days after delivery of either the transgene or PBS, animals were reanesthetized and mechanically ventilated. A clamshell thoracic incision was performed before 3000 IU of heparin was injected intravenously. The inferior vena cava was transected, and animals were partially exsanguinated to unload the LV before the aorta was cross-clamped and 30 mL of University of Wisconsin cardioplegic solution was injected into the LV cavity, allowing cardiac arrest within 5 seconds. The great vessels, pulmonary veins, and superior vena cava were transected, and the heart was transferred into 0.9% saline solution at 4°C. After 15 minutes or 4 hours, hearts were hung on a modified Langendorff apparatus and perfused as previously described.19,20 An LV latex balloon was positioned and connected to a pressure transducer (Millar Instruments), and its volume was adjusted to assess a baseline condition of 0 mm Hg end-diastolic pressure (LVEDP). After 30 minutes of reperfusion, baseline LV pressures, responses to standard increases of end-diastolic volume (LVEDV), and responses to standard doses of isoproterenol (Iso) were recorded. After termination of functional measurements, hearts were kept perfused for 30 minutes before samples of the ventricles were frozen in liquid nitrogen for biochemical analysis. A control group included hearts isolated from rabbits that had not undergone surgery, quickly harvested without being arrested, and immediately reperfused on the Langendorff apparatus.
Myocardial ßAR Density and Signaling
Membranes were prepared as previously described.15,17,21 Total ßAR density was determined by radioligand binding with a saturating concentration (300 pmol/L) of 125I-labeled cyanopindolol at 37°C for 1 hour as described.17 For adenylyl cyclase activity, 20 µg of myocardial membrane protein was incubated with 0.1 µmol/L [
-32P]ATP for 15 minutes at 37°C under basal conditions or in the presence of 1 mmol/L Iso or 10 mmol/L sodium fluoride (NaF). cAMP production was quantified by standard methods described previously.15,17 GRK activity assay was assessed in cytosolic or membrane fractions with rhodopsin-enriched rod outer segment membranes as we have previously described.10 ßARK1 levels were visualized by Western blotting as previously described.10
Northern Analysis
Total RNA was isolated from frozen LV samples, and Northern blot analysis was performed by standard methods as previously described.17
Statistical Analysis
Data are expressed as mean±SEM. Student’s t test was used for comparison between groups, whereas comparison of Iso dose-response or LVEDV response were done by 2-way ANOVA. For all tests, a value of P<0.05 was considered significant.
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Results |
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LV Physiology After Cardioplegia
To examine the effect of ßARK1 inhibition on postcardioplegic LV dysfunction, we delivered either the ßARKct transgene (Adv-ßARKct) or PBS into normal rabbit hearts by use of an intracoronary in vivo delivery technique we recently developed.18 Transgene expression was confirmed by Northern blot analysis 5 days after delivery of 5x1011 TVP of Adv-ßARKct (n=9) (Figure 1). LV contractility (LV dP/dtmax), LV relaxation (LV dP/dtmin), and LVEDP were all progressively altered by prolonged cardioplegic arrest in hearts that received only PBS compared with nonarrested hearts (Table and Figure 2). In particular, the response to Iso was significantly decreased after 4 hours of cardioplegic arrest compared with control nonarrested hearts (Figure 2C and 2D). LV contractility and relaxation in hearts treated with Adv-ßARKct 5 days before surgery, however, were both significantly increased during the reperfusion period after a 15-minute cardioplegic arrest compared with PBS-treated arrested hearts (Figure 2A and 2B). LV function was also improved after 4 hours of cardioplegic arrest in hearts that received intracoronary Adv-ßARKct delivery 5 days earlier as opposed to hearts that received PBS only (Figure 2C and 2D). In fact, in the more immediate cardioplegic setting (15 minutes), ßARKct expression even enhanced the LV function of hearts significantly above that of the nonarrested control hearts (Figure 2A and 2B). Interestingly, in Adv-ßARKct–treated hearts after 4 hours of cold cardioplegia, LV function was still comparable to that of normal nonarrested hearts (Figure 2C and 2D).
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; A and B, n=5; C and D, n=4) or PBS (
; A through D, n=4). Nonarrested control hearts were directly perfused on Langendorff apparatus (
; A through D, n=4). For statistical comparison, complete response curves were compared between groups by 2-way ANOVA. Thus, overall change in these cardiac functional parameters could be assessed. Our statistical findings were as follows: A, *P<0.005 (Adv-ßARKct vs PBS), 
P<0.05 (Adv-ßARKct vs control). B, *P<0.005 (Adv-ßARKct vs PBS), 
P=NS (Adv-ßARKct vs control). D, *P<0.005 (Adv-ßARKct vs PBS), In addition, LV compliance, as measured by LVEDP variation with increasing preload, was significantly impaired after 4 hours of cardioplegic arrest. This parameter of LV function, however, was also restored with ßARKct expression, because LVEDP remained in the normal range for hearts previously treated with Adv-ßARKct (Figure 3). Increased heart rate can sometimes occur after cardiac insult and thus be a sign of cardiac dysfunction. We found significantly increased heart rate at 4 hours after CPB (Figure 4). Importantly, all groups that had previously been treated with intracoronary ßARKct had normal heart rates compared with control nonarrested hearts (Figure 4).
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ßAR Signaling After Cardioplegic Arrest
We analyzed biochemical ßAR signaling in myocardium after reperfusion of arrested hearts. This was done 30 minutes after termination of functional measurements to allow washout of residual Iso. After 15 minutes of cardioplegic exposure, myocardial ßAR density was already decreased in LV membranes prepared from hearts that received PBS 5 days earlier (Figure 5A). A similar loss of ßAR density was also evident in hearts arrested for 4 hours (Figure 5A). Hearts that received Adv-ßARKct 5 days previously, however, had significantly higher ßAR density at either 15 minutes or 4 hours, and these values were in the normal range compared with control hearts not exposed to cardioplegia (Figure 5A).
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P<0.05 (PBS 4 hours vs control). B, GRK activity expressed as percent of values of control hearts. *P<0.05 (PBS 4 hours vs PBS 15 minutes),
These acute changes in ßAR density may reflect internalization, and that may include hyperactive desensitization mechanisms. Therefore, we measured ßARK1 (GRK2) levels and activity. We have previously shown that cytosolic myocardial GRK activity is almost exclusively a result of ßARK1.22 We found GRK activity to be significantly increased after 4 hours of cardioplegia in hearts previously treated with PBS (Figure 5B). GRK activity, however, was unaltered even after prolonged cardioplegic arrest in hearts treated with Adv-ßARKct (Figure 5B). Because active ßARK1 resides in the membrane fraction of intact cells after Gß-dependent translocation, however, we also examined the levels of membrane ßARK1 by immunoblotting. ßARK1 levels were elevated in membranes from PBS-treated arrested hearts (Figure 5C). In contrast, less ßARK1 was found in the membrane of ßARKct-expressing hearts in both the short and long term (Figure 5C).
We also examined ßAR signaling in these hearts. Basal and Iso-stimulated adenylyl cyclase activity were decreased after 15 minutes of cold cardioplegic arrest compared with normal nonarrested hearts, whereas they remained unchanged in Adv-ßARKct hearts (Figure 5D). NaF-stimulated adenylyl cyclase activity was unchanged at 15 minutes but decreased after 4 hours of cardioplegic exposure (Figure 5D). This may indicate postreceptor defects after cardiac arrest. Interestingly, in LV membrane prepared from hearts that received Adv-ßARKct 5 days earlier, NaF-stimulated adenylyl cyclase activity was unchanged at both 15 minutes and 4 hours (Figure 5D). Thus, ßARKct expression was capable of restoring the myocardial ßAR signaling system in cardioplegia-arrested hearts.
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Discussion |
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Inhibition of ßARK1 activity by adenovirus-mediated gene transfer of the ßARKct 5 days before a cold cardioplegic cardiac arrest prevents myocardial dysfunction when the heart is reperfused. Compared with normal nonarrested hearts, LV function in ßARKct-expressing hearts is improved after a short cold ischemic period, and although function progressively decreases with prolonged cardiac arrest, ßARKct expression maintains function within normal limits and delays development of the functional consequences of cardioplegic injury. Previous studies examining biochemical abnormalities of cold cardioplegia or warm blood reperfusion suggested that defects in ßAR signaling were associated with this treatment.2–6 Notably, early changes in ßAR coupling with its effector adenylyl cyclase occur, similar to what is observed during evolution of chronic cardiac failure.2–5 Our present results demonstrate that gene transfer of a ßARK1 inhibitor peptide prevents alterations in ßAR signaling and therefore confirm a role of ßARK1 in altering ventricular function after cardioplegic arrest.
We have previously demonstrated the positive effect of ßARKct overexpression on myocardial function in vivo in transgenic mice15,23,24 or ex vivo in transplantation studies in which transgenes were delivered immediately after graft harvest and before transplantation of the graft into the recipient animal.20 In addition, we recently delivered Adv-ßARKct in rabbits simultaneously with the creation of an LV myocardial infarction and observed improved LV function compared with animals that received an empty virus or PBS only.17 In this study, ßAR signaling remained normal in animals treated with the ßARK inhibitor transgene, whereas it was dramatically impaired in nontreated animals, with a reduction in ßAR density, a decrease in adenylyl cyclase activity in response to Iso stimulation, and increased expression and activity of ßARK1.17 In fact, the development of heart failure that initially is associated with myocardial infarction in this model was significantly delayed in the presence of the ßARKct transgene, demonstrating that ßARK1 is critically involved in the pathogenesis of ischemic cardiomyopathy.17
The design of our present study offers insight into the acute functional and biochemical changes that occur during the reperfusion period after cardioplegic arrest. Modifications of ßAR signaling were dependent on the exposure time to cardioplegic solution in previously untreated (PBS) animals, whereas no significant changes were observed in animals that were pretreated with Adv-ßARKct. This effect was seen in hearts exposed short-term or for as long as 4 hours. The immediate loss in ßAR density, which persisted after prolonged exposure to cardioplegic solution, was abolished in hearts expressing the ßARKct. In fact, ßAR density was significantly higher than control counterparts in right ventricles of ßARKct-treated hearts, whereas it was unchanged in PBS-treated hearts (data not shown). This suggests that downregulation as opposed to desensitization of ßARs is probably not a critical event in the sequence of cardioprotective adaptive mechanisms. The observation that chronic ßAR antagonist administration in patients before CPB with cardioplegic arrest did not prevent acute desensitization supports this hypothesis.3,4
With respect to desensitization, we found increased activated ßARK1 levels in arrested hearts. Importantly, in the ßARKct-treated hearts, less ßARK1 appeared to be actively translocated from the cytosol to the membrane after cardioplegia and reperfusion. Thus, it appears that ßARK1 plays a critical role in the ßAR changes associated with cardioplegic arrest and thus acts as the primary target for ßARKct action. Therefore, dynamic changes that occur progressively during cold myocardial ischemia certainly explain, at least partially, the progressive degradation in LV function with prolonged cardioplegic cardiac arrest. Adenovirus-mediated gene transfer of ßARKct appears to stabilize ßAR signaling during cardioplegia and reperfusion and consequently improves LV contractility and relaxation, as well as LV compliance and heart rate.
Adenylyl cyclase activity in response to ß-agonist stimulation is clearly affected by cold cardioplegic exposure, as demonstrated by animal studies2 as well as results obtained from human right atrial samples taken during CPB procedures.3–6 Few studies have analyzed the adenylyl cyclase activity in response to NaF stimulation, and results are controversial. Some studies show no changes in direct G protein–stimulated adenylyl cyclase activity,5 whereas others demonstrate a decreased activity in animals6 or in human samples.3 Our results demonstrate a time-dependent variation in NaF-stimulated adenylyl cyclase activity. Although Iso-stimulated adenylyl cyclase activity was already decreased after a short period of cardioplegic arrest, direct adenylyl cyclase stimulation via G proteins (NaF) showed normal activity after 15 minutes of cardioplegia, whereas it was significantly decreased after prolonged exposure to cardioplegia. Thus, with prolonged cardiac arrest, there were both receptor and postreceptor defects in this system. Gene transfer of ßARKct before cardioplegic cardiac arrest, however, preserved the adenylyl cyclase pathway at all levels. This is probably due to the overall improved function before cardioplegic arrest.
As opposed to progressive modifications in ßAR signaling during development of chronic heart failure, CPB represents a clinically relevant situation in which early alterations in ßAR signaling take place as a consequence of changes in the extracellular milieu during both the cardioplegia and reperfusion periods. Cold cardioplegic solutions are used daily, not only for preservation of a donor heart during harvesting and transport but also primarily for myocardial protection during CPB and cardiac arrest. Ventricular dysfunction that occurs at the time of reperfusion prolongs any hospital stay and favors postcardiotomy morbidity. Therefore, there is a clinical need for different cardioplegic methods that not only provide myocardial protection during the cold ischemic period but also mainly ensure adequate ventricular function during reperfusion. In this regard, adenovirus-mediated gene transfer of an inhibitor of ßARK1 before cold cardioplegic arrest may constitute a new strategy to prevent postoperative LV dysfunction. Moreover, it is possible that small molecules could be developed to inhibit ßARK1 activity in a pharmacological manner, which may represent a novel therapeutic approach for acute cardiac dysfunction.
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Acknowledgments |
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This work was supported by a grant from the Swiss National Science Foundation (Dr Tevaearai) and NIH grants HL-59533 (Dr Koch) and HL-56205 (Dr Koch). The authors thank Dr Robert J. Lefkowitz, MD, for helpful discussion; K. Campbell, Dr Janelle R. Keys, and Dr J. Kurt Chapman for excellent technical assistance; and the Genzyme Corp (Framingham, Mass) for preparation and purification of Adv-ßARKct.
Received May 15, 2001; revision received July 26, 2001; accepted July 30, 2001.
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