Published online before print March 29, 2004, doi: 10.1161/01.CIR.0000125521.40985.28
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(Circulation. 2004;109:1590-1593.)
© 2004 American Heart Association, Inc.
Brief Rapid Communication |
Targeted ß-Adrenergic Receptor Kinase (ßARK1) Inhibition by Gene Transfer in Failing Human Hearts
From the Department of Surgery, Duke University Medical Center (M.L.W., J.A.H., J.S., E.R., J.P., A.J., C.A.M., W.J.K.), Durham, NC; Department of Surgery, Massachusetts General Hospital (M.L.W.), Boston, Mass; and Center for Translational Medicine (W.J.K.), Thomas Jefferson University, Philadelphia, Pa.
Correspondence to Walter J. Koch, PhD, FAHA, Director, Center for Translational Medicine, Jefferson Medical College, 1025 Walnut St, Room 410, Philadelphia, PA 19107. E-mail walter.koch{at}jefferson.edu
Received December 16, 2003; revision received February 12, 2004; accepted February 18, 2004.
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Abstract |
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Background— Failing human myocardium is characterized by an attenuated contractile response to ß-adrenergic receptor (ßAR) stimulation due to changes in this signaling cascade, including increased expression and activity of the ß-adrenergic receptor kinase (ßARK1). This leads to desensitization and downregulation of ßARs. Previously, expression of a peptide inhibitor of ßARK1 (ßARKct) has proven beneficial in several animal models of heart failure (HF).
Methods and Results— To test the hypothesis that inhibition of ßARK1 could improve ß-adrenergic signaling and contractile function in failing human myocytes, the ßARKct was expressed via adenovirus-mediated (AdßARKct) gene transfer in ventricular myocytes isolated from hearts explanted from 10 patients with end-stage HF undergoing cardiac transplantation. AdßARKct also contained the marker gene, green fluorescent protein, and successful gene transfer was confirmed via fluorescence and immunoblotting. Compared with uninfected failing myocytes (control), the velocities of both contraction and relaxation in the AdßARKct-treated cells were increased in response to the ß-agonist isoproterenol (contraction: 57.5±6.6% versus 37.0±4.2% shortening per second, P<0.05; relaxation: 43.8±5.5% versus 27.5±3.9% lengthening per second, P<0.05). Fractional shortening was similarly enhanced (12.2±1.2% versus 8.0±0.9%, P<0.05). Finally, adenylyl cyclase activity in response to isoproterenol was also increased in AdßARKct-treated myocytes.
Conclusions— These results demonstrate that as in animal models of HF, expression of the ßARKct can improve contractile function and ß-adrenergic responsiveness in failing human myocytes. Thus, ßARK1 inhibition may represent a therapeutic strategy for human HF.
Key Words: myocytes • heart failure • gene therapy
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Introduction |
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Failing human myocardium is characterized by a decreased responsiveness to ß-adrenergic stimulation.1 This is attributed to a reduction in the myocardial density of ß-adrenergic receptors (ßARs) and functional uncoupling of the ßAR pathway.2 ßARs, like other G-protein–coupled receptors (GPCRs), undergo desensitization and downregulation in response to ongoing stimulation. GPCR kinases (GRKs) phosphorylate activated receptors, which leads to an incapacity for further G-protein stimulation.3 For ßARs, the most important GRK appears to be GRK2, or ßARK1, which desensitizes ßARs and other GPCRs via membrane translocation dependent on direct binding to dissociated ß
-subunits of G proteins (Gß).4 In human heart failure (HF), the expression and activity of ßARK1 are elevated, which contributes to the lack of ß-adrenergic reserve.5 This is probably the result of enhanced sympathetic nervous system activity and excessive catecholamine stimulation of ßARs associated with the failing heart.6
It has been demonstrated in numerous animal models that inhibition of ßARK1 translocation with a peptide containing the Gß-binding site (ßARKct) can lead to in vivo ßARK1 inhibition and rescue of HF.3,4 Cardiac expression of the ßARKct, whether in transgenic mice or after intracoronary gene delivery to larger animal models of HF, can improve myocardial function and lead to enhanced responsiveness to catecholamines through preservation of ßAR density and G-protein coupling.7–12
Currently, it is unknown whether expression of the ßARKct peptide will lead to improvement of function in the failing human heart. Because myocytes from failing human hearts are characterized by an attenuated response to ßAR stimulation and contractile dysfunction,13,14 we sought to determine, as an ultimate "proof of concept" for ßARK1 inhibition for HF therapy, whether delivery of the ßARKct to failing human myocytes via adenovirus-mediated gene transfer would result in enhanced function and ßAR signaling.
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Methods |
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Human Myocyte Isolation and Gene Transfer
All experiments were performed in accordance with a protocol approved by the Duke University Institutional Review Board. Failing human myocytes were isolated from 10 different hearts explanted from patients undergoing transplantation by a previously reported method.15 Myocytes were then infected with an adenovirus coexpressing ßARKct and the indicator protein GFP (green fluorescent protein) driven by separate cytomegalovirus promoters or an adenovirus expressing GFP alone at a multiplicity of infection of 100:1. Another control group consisted of myocytes that remained uninfected. Twenty-four hours after infection, myocytes were suspended in a Krebs solution with 2 mmol/L Ca2+. Contractile function of rod-shaped cells (80 to 200 µm) under basal and isoproterenol (ISO)-stimulated (10–6 mol/L) conditions was measured during electrical field stimulation with a video edge-detection system (Crescent Electronics). A mean of 9.3±0.5 cells were analyzed per condition per heart.
Adenylyl Cyclase Activity
Adenylyl cyclase assays were performed with [3H]-labeling in a cAMP assay system (Amersham Biosciences; cells from n =5 patients). Analysis was performed 36 hours after myocyte isolation under basal, ISO-stimulated (10–6 mol/L), and forskolin-stimulated (10–6 mol/L) conditions as described previously.16
Transgene Expression
ßARKct transgene expression was confirmed in whole-cell lysates with an anti-GRK2 antibody (Santa Cruz Biotechnology) at a dilution of 1:2500 as described previously,9 and GFP expression was confirmed by fluorescence.
Statistical Analysis
Contractility data were averaged for each group and condition with each heart, and each heart was treated as a single data point. One-way ANOVA was used for testing for differences among multiple groups; Bonferroni or Fisher’s least significant difference tests were used for post hoc testing.
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Results |
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Myocytes were isolated from 10 hearts explanted from patients undergoing cardiac transplantation. Twenty-four hours after infection, 100% of rod-shaped myocytes expressed the GFP transgene, and expression of the ßARKct transgene was confirmed by immunoblotting (Figure 1).
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Representative tracings of failing and ßARKct-expressing myocyte contractility are shown in Figure 2A, with functional data shown in Figure 2B for the experimental groups. AdßARKct-treated myocytes demonstrated enhanced contractile function in response to ßAR stimulation. At baseline, there was no difference between uninfected and ßARKct-treated myocytes in velocity of contraction (21.1±3.4% versus 28.0±4.7%) or relaxation (21.2±3.0% versus 28.0±4.8%; Figure 2B). With ISO stimulation, failing myocytes treated with AdßARKct demonstrated a significant increase in velocity of both contraction (57.5±6.6% versus 37.0±4.2%, P<0.05) and relaxation (43.8±5.5% versus 27.5±3.9%, P<0.05). ISO-stimulated fractional shortening of AdßARKct-infected myocytes was higher than control cells (12.2±1.2% versus 8.0±0.9%, P<0.05). In myocytes isolated from 5 hearts, an additional control was used because cells were infected with a virus that contained only GFP. Contractile results from these failing myocytes were statistically identical to those from uninfected myocytes (Figure 2C).
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P<0.05 by ANOVA and Fisher’s least significant difference test. C, % Fractional shortening in uninfected failing human myocytes and control myocytes infected with AdGFP (n=5). D, Adenylyl cyclase (AC) activity after cAMP production and in response to ISO is enhanced after inhibition of ßARK1(n=5); *P<0.05 by Student’s t test.Figure 2D depicts ISO-stimulated adenylyl cyclase activity and cAMP production normalized to forskolin-stimulated levels. cAMP production in response to ßAR stimulation was increased significantly in the AdßARKct-treated myocytes compared with uninfected cells (37.2±5.9% versus 21.4±1.4%, P<0.05).
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Discussion |
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The procurement of functional failing human myocytes is difficult, and gene transfer studies are limited. The method of myocyte isolation used in the present study yielded a relatively large number of cells from each heart and allowed repeated measurements. Moreover, the use of total patient numbers (n=10) instead of cell number allows for more rigorous statistical testing of the potential effects of a transgene on failing myocyte function. Previously, only 2 studies have been published using adenovirus gene transfer to target abnormal Ca2+ handling in the failing human myocyte.17,18 The present study demonstrates for the first time that inhibition of ßARK1 activity in failing human myocytes can improve ßAR responsiveness and contractile function.
The upregulation of ßARK1 in HF5 appears to be maladaptive, because correction of this abnormality by expression of the ßARKct via gene delivery in animal models can maintain density and coupling of ßARs, thus restoring responsiveness and contractile function.9,12 In the present study, we sought to determine whether the salutary effects of the ßARKct transgene would extend to failing human myocytes. Our results demonstrate an ultimate "proof of concept," because ßARK1 inhibition can increase cAMP production in response to ßAR stimulation and significantly improve the contractile response of the failing human myocyte. One limitation of the present study is the absence of nonfailing myocytes. However, our myocyte purification technique utilizes the intact heart, and we are unable to obtain nonfailing cells in any meaningful amount or frequency.
Although it may appear that the inhibition of ßARK1 in failing myocytes contradicts a proven treatment of HF (ß-blockade), at a molecular level, this is not the case. It has been demonstrated that catecholamine stimulation increases ßARK1 expression, whereas ßAR-blockade has the opposite effect.6 Thus, both ßARKct expression and ß-blocker treatment can decrease GRK activity in the heart and preserve membrane density of ßARs, restoring the integrity of the ß-adrenergic system in HF.3 Importantly, in a murine HF model, treatment with the ß-blocker metoprolol potentiated the effects of the ßARKct.11 Therefore, these treatments appear to act synergistically (and not in an opposing manner) to improve the contractile performance of the failing heart during periods of ßAR stimulation. Six of 10 HF patients studied were receiving ß-blockers at the time of transplantation. Despite the possibility that these hearts may have had reduced ßARK1 levels, significant improvement in function was observed in these myocytes treated with the ßARKct. Moreover, enhanced contractile performance was seen regardless of the status of ß-blockade.
Because the ßARKct mechanism involves blocking the Gß-mediated activation of ßARK1, the beneficial effects of this peptide may include enhanced signaling through other GPCRs that are targets for this GRK. Moreover, there may be inhibition of other as yet unidentified Gß effectors. These alternative mechanisms have been discussed previously.3
Gene therapy remains an important area of investigation for the future of HF therapy. Despite significant research efforts, the outlook for patients with end-stage HF remains grim. It is our hope that further research of potential targets for HF and improved gene-delivery technology will advance gene therapy as a viable option for these HF patients. Because our results demonstrate that, as in previous animal studies, ßARK1 inhibition can improve the ßAR signaling and contractile function of failing human myocytes, delivery of the ßARKct may emerge as a novel treatment for HF.
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Acknowledgments |
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The authors thank Genzyme Corporation for purification of some of the AdßARKct vector. This study was supported by National Institutes of Health grants R01 HL56205 and HL59533 (to Dr Koch) and R01 HL072183 (to Dr Milano).
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Footnotes |
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*Drs Williams and Hata contributed equally to this project.
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References |
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