Published online before print September 28, 2006, doi: 10.1161/01.RES.0000247932.71270.2c
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© 2006 American Heart Association, Inc.
Integrative Physiology |
Cardiac-Specific Ablation of G-Protein Receptor Kinase 2 Redefines Its Roles in Heart Development and ß-Adrenergic Signaling
From the Center for Molecular Cardiovascular Research (S.J.M., A.D., J.L.K., D.J.H., Y.M., A.M.O., E.W.B., G.W.D.), University of Cincinnati, Ohio; Center for Translational Medicine (W.J.K.), Jefferson Medical College, Philadelphia, Pa; and Institute of Biosciences and Technology (R.J.S.), Texas A&M University Health Science Center, Houston.
Correspondence to Gerald W Dorn II, MD, Hanna Professor and Director, Molecular Cardiovascular Research, 231 Albert Sabin Way, ML 0839, Cincinnati, OH 45267-0839. E-mail dorngw{at}ucmail.uc.edu
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Abstract |
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G-protein receptor kinase 2 (GRK2) is 1 of 7 mammalian GRKs that phosphorylate ligand-bound 7-transmembrane receptors, causing receptor uncoupling from G proteins and potentially activating non–G-protein signaling pathways. GRK2 is unique among members of the GRK family in that its genetic ablation causes embryonic lethality. Cardiac abnormalities in GRK2 null embryos implicated GRK2 in cardiac development but prevented studies of the knockout phenotype in adult hearts. Here, we created GRK2-loxP–targeted mice and used Cre recombination to generate germline and cardiac-specific GRK2 knockouts. GRK2 deletion in the preimplantation embryo with EIIa-Cre (germline null) resulted in developmental retardation and embryonic lethality between embryonic day 10.5 (E10.5) and E11.5. At E9.5, cardiac myocyte specification and cardiac looping were normal, but ventricular development was delayed. Cardiomyocyte-specific ablation of GRK2 in the embryo with Nkx2.5-driven Cre (cardiac-specific GRK2 knockout) produced viable mice with normal heart structure, function, and cardiac gene expression. Cardiac-specific GRK2 knockout mice exhibited enhanced inotropic sensitivity to the ß-adrenergic receptor agonist isoproterenol, with impairment of normal inotropic and lusitropic tachyphylaxis, and exhibited accelerated development of catecholamine toxicity with chronic isoproterenol treatment. These findings show that cardiomyocyte autonomous GRK2 is not essential for myocardial development after cardiac specification, suggesting that embryonic developmental abnormalities may be attributable to extracardiac effects of GRK2 ablation. In the adult heart, cardiac GRK2 is a major factor regulating inotropic and lusitropic tachyphylaxis to ß-adrenergic agonist, which likely contributes to its protective effects in catecholamine cardiomyopathy.
Key Words: ß-adrenergic receptor • G protein–coupled receptor kinase • recombination • contractility • isoproterenol • desensitization
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Introduction |
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G-protein receptor kinases (GRKs) function as "off switches" for the superfamily of G-protein coupled, 7-transmembrane spanning receptors (7TMR), members of which are activated by such diverse stimuli as photons, odorants, neurotransmitters, and cardio-active hormones.1,2 Receptor phosphorylation by any of the 7 mammalian GRKs induces recruitment and binding of ß-arrestin, which displaces bound G proteins and uncouples the receptor from downstream signaling effectors in a process termed "desensitization." Additionally, ß-arrestin targets receptors to clathrin-coated pits for endocytic internalization, resulting in receptor downregulation.3 There is accumulating evidence that, coincident with termination of G-protein signaling, GRK phosphorylation of 7TMR can activate parallel signaling pathways involving nonreceptor tyrosine kinases or small GTP binding proteins.4 Here, ß-arrestin recruitment to GRK-phosphorylated 7TMRs creates a scaffold for macromolecular complexes of kinases that transduce nonclassical signaling events.
The multiplicity of GRKs is associated with specific roles for each, related to differential selectivity for individual 7TMR and distinct spatiotemporal expression patterns. Gene ablation studies have been especially valuable in defining the essential functions of individual GRKs by generating phenotypes related to impaired desensitization of specific receptor types. Accordingly, deletion of retinal GRK1 caused persistence of visual signals,5 GRK3 ablation affected sensing of odors,6 GRK5 ablation produced an asthma-like phenotype and behavioral supersensitivity related to persistent cholinergic signaling,7,8 and GRK6 ablation caused hypersensitivity to cocaine and amphetamines because of impaired desensitization of central nervous system dopamine receptors.9 Strikingly, ablation of the gene for GRK2, also known as ß-adrenergic receptor (ß-AR) kinase 1,10 was unique in causing intrauterine heart failure and death.11 Embryonic lethality and cardiovascular defects specific to GRK2 ablation suggested that a specialized function exists for this GRK (relative to other GRK family members) in fetal development and/or cardiac morphogenesis.
GRK2 is highly expressed in myocardium, where by phosphorylating ß-ARs it regulates cardiac contractility.12 Multiple lines of evidence suggest that GRK2 may also contribute to the pathology of heart failure: myocardial GRK activity is increased in human heart failure13; GRK2 levels are increased in circulating lymphocytes of hypertensive patients14; and GRK2 is upregulated in experimental myocardial ischemia, in which it is temporally associated with diminished ß2-AR responsiveness.15 Furthermore, inhibition of GRK2 by myocardial expression of a C-terminal peptide, ßARKct, can restore ß-AR signaling and contractile function in pressure overload hypertrophy,16 dilated cardiomyopathy,17 and ischemia/infarction models.18 Thus, GRK2 appears to be an important regulator of cardiac function in health and disease.
Here, we have generated mice in which the GRK2 gene can be conditionally ablated using Cre-lox technology. We find that germline ablation of GRK2 results in embryonic lethality from embryonic day 10.5 (E10.5), associated with a generalized delay in embryonic development. In contrast, cardiomyocyte-specific ablation of GRK2 in the embryonic heart using Nkx2.5-directed Cre expression results in viable mice with normal heart development but impaired inotropic and lusitropic tachyphylaxis during sustained ß-adrenergic stimulation and pronounced aggravation of catecholamine toxicity following chronic administration of ß-adrenergic agonist.
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Materials and Methods |
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Mouse Generation
Exons 3 to 6 of GRK2 were targeted by flanking them with loxP sites, in combination with a frt-flanked neomycin phosphotransferase module to positively select putative homologous recombinant ES cells. Correctly targeted recombinants were identified via XbaI restriction digest and Southern blot with a 5' probe external to the targeting vector. Following implantation of ES cells into blastocysts, generation of chimeric mice, and breeding to the F1 generation, heterozygous GRK2-targeted mice were bred with Flp transgenic mice19 to delete the neomycin selection cassette. Progeny were crossed to remove the Flp transgene, resulting in mice bearing floxed GRK2 alleles (heterozygous GRK2f/+; homozygous GRK2f/f). EIIa-Cre transgenic mice20 were bred on to the GRK2f/+ background and crossed with GRK2f/f female mice to generate GRK2 null embryos. Nkx2.5-Cre knock-in mice21 were bred on to the GRK2f/f background to generate cardiac GRK2-deleted (GRK2del) mice, GRK2f/f;Nkx2.5Cre. GRK2f/f and GRK2f/f;Nkx2.5Cre mice were maintained on a mixed 129/C57BL6 genetic background. Mice were housed according to procedures approved by the University of Cincinnati Institutional Animal Care and Use Committee.
Embryonic Studies
Embryos were harvested from timed pregnancies, frozen or fixed in 4% paraformaldehyde, and genotyped by PCR. Histological studies and whole-mount in situ hybridization used standard techniques.22 Digoxygenin-labeled Nkx2.5 riboprobe was produced with a kit from Boehringer Mannheim (no. 1175-025) and visualized using alkaline phosphatase.
Analysis of Gene Recombination
For quantitative PCR (qPCR) of floxed and recombined (deleted) GRK2 alleles in embryonic and adult hearts, DNA was extracted by proteinase K digestion, phenol:chloroform:isoamyl alcohol purification, and ethanol precipitation. Genomic DNA (100 ng) was amplified with Bio-Rad iQ Supermix containing iTaq DNA polymerase and SYBR Green I, and fluorescence was monitored on an Opticon 2 real-time PCR instrument (Bio-Rad/MJ Research). Amplification of floxed GRK2 alleles was performed using 2 ng/µL oligos A (5'-CAGGCATTCCTGCTGGACTAG-3') and B (5'-TGAGGCTCAGGGATACCTGTCAT-3'), whereas amplification of recombined (deleted) GRK2 alleles was performed using oligos A and C (5'-GTTAGCTCAGGCCAACAAGCC-3') (see Figure 2 for positions of PCR primers), using cycling parameters of 94°C, 5 minutes, followed by 35 cycles of 94°C, 30 seconds, 60°C, 1 minute, and 72°C, 40 seconds. A melting curve with continuous fluorescence monitoring was performed following qPCR to verify that only 1 PCR amplicon was detectable, and the final product obtained from qPCR amplification was further analyzed on standard ethidium bromide (EtBr)-stained agarose gels to confirm its size. The critical threshold fluorescence level, C(t), for each qPCR was defined as equal to 10x the SD of the fluorescence signal obtained between cycles 3 to 8, after fluorescence had been corrected for signal obtained from blank wells (without genomic DNA). Baseline fluorescence (defined as the average fluorescence over cycles 3 to 8) was subtracted from sample fluorescence. Results were calculated as 
C(t)[GRK2del– GRK2flox] for each genomic DNA sample. The percentage of recombination in each sample is equal to 100x(1/[1+2C(t)[GRK2del–GRK2flox]]). As examples, a C(t) value of 0 indicates GRK2 heterozygosity, or 50% recombination (assuming equal amplification efficiency of GRK2flox and GRK2del), whereas a 1:3 ratio of floxed to deleted alleles (75% recombination) results in C(t)[GRK2del–GRK2flox]=–1.6.
For analysis of GRK2 recombination in individual cardiomyocytes, GRK2f/f and GRK2f/f;Nkx2.5-Cre mouse hearts underwent collagenase digestion; single rod-shaped cardiomyocytes were identified by phase-contrast microscopy and transferred to 3.5 µL of PBS. Whole-genome amplification of DNA from each individual cardiomyocyte was performed using the Qiagen REPLI-g Mini Kit. Briefly, 3.5 µL of buffer D2 (containing 0.08 mol/L dithiothreitol) was added to 3.5 µL of PBS containing a single cardiomyocyte, incubated on ice for 10 minutes, and terminated with 3.5 µL of Qiagen Stop Solution. Genomic DNA was amplified at 30°C for 16 hours using Qiagen REPLI-g Mini polymerase according to the protocol of the manufacturer. Half of the amplified product was extracted with phenol:chloroform:isoamyl alcohol; the DNA was precipitated and used as template for conventional PCR genotyping.
Immunoblot Analyses
Tissue homogenates underwent immunoblotting for GRK2 and GRK5 content using standard techniques and Santa Cruz sc-562 anti-GRK2 or sc-565 anti-GRK5, with chemiluminescence detection. For measurements of isoproterenol-stimulated receptor phosphorylation, isolated Langendorff-cannulated hearts were perfused with buffer (control) or 1 µmol/L isoproterenol for increasing periods of time, as indicated, and the hearts instantly frozen using liquid nitrogen-cooled Wallenberg tongs. Clarified cardiac homogenates prepared from frozen hearts were used for immunoblot detection using total and S355/S356 phospho-specific human ß2-AR antibodies from Cell Signaling.
Cardiac Functional Studies
Echocardiography in nonsedated mice, closed chest invasive hemodynamic studies, and RNA dot blotting were performed using standard techniques.23 For ß-AR desensitization, the hemodynamic response to increasing doses of intravenous isoproterenol was determined, followed immediately by a continuous high-dose infusion sustained for 30 minutes. Isoproterenol was chronically infused using subcutaneously implanted Alzet osmotic mini-pumps
Statistical Analysis
Cardiac contractility data were analyzed using 2-sided Student’s t test for comparison of 2 groups, or 1-way ANOVA for analysis of more than 2 groups, with Tukey’s post hoc comparison. Significance was taken at P<0.05.
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Retarded Development and Embryonic Lethality in Germline GRK2 Null Mice
To further evaluate the function of GRK2, we generated a mouse line in which exons 3 through 6 are flanked by 2 loxP sites (GRK2 floxed mice, GRK2f/f) (Figure 1A). Crossing this mouse line for 2 generations with mice that express Cre recombinase under control of regulatory sequences for zygotically expressed adenovirus EIIa gene20 generated germline deletion of GRK2 (GRK2 null mice; GRK2f/f;EIIaCre mice). From heterozygous crosses, no homozygous mutants were born (21/62 +/+, 41/62 +/–, 0/62 –/–), indicating embryonic lethality (Figure 1B). Litters from heterozygous crosses at E9.5 exhibited normal Mendelian inheritance of the mutant allele (18/66 +/+, 32/66 +/–, 16/66 –/–) (Figure 1C, top), whereas E10.5 embryos showed fewer than expected homozygous mutants (13/39 +/+, 18/39 +/–, 8/39 –/–), and E11.5 litters showed no homozygous mutants of 21 viable embryos. GRK2 protein was not detectable in E9.5 GRK2 null mice (Figure 1C, bottom). These data indicate that GRK2 null mice die between E10.5 and E11.5. At E10.5, GRK2-deficient embryos exhibited growth retardation compared with wild-type (GRK2f/f) embryos (Figure 1D). Comparison of littermates from E7.5 through E10.5 indicated that growth arrest in GRK2 null embryos occurred at E8.5 to E9.0. Cardiac looping was normal, as was expression of the apical cardiac regulatory gene Nkx2.5 (Figure 1E). Histological examination of the heart revealed hypoplasia of the single ventricle (Figure 1F). The ventricular muscle was only 1-cell thick, and some GRK2 null hearts had pericardial edema, suggesting embryonic heart failure (Figure 1F). No phenotypic abnormalities were detected in GRK2 heterozygotes (data not shown), suggesting that there is a threshold level of GRK2 gene dosage below which embryogenesis is impaired.
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Cardiomyocyte GRK2 Is Not Essential for Myocardial Development and Embryonic Viability
The cardiac phenotype of germline GRK2 null embryos revealed impaired or delayed heart development. We considered that this could be either a cause of embryonic growth retardation, or a consequence of a more general or noncardiac effect on embryonic development.24 To address this issue, we ablated GRK2 specifically in embryonic cardiac myocytes by breeding GRK2f/f mice to mice expressing Cre recombinase under control of the Nkx2.5 gene promoter21 (cardiac GRK2del mice; GRK2f/f;Nkx2.5Cre) (Figure 2A). Cardiac GRK2del mice were viable, with normal Mendelian inheritance of the mutant gene (Figure 2B). GRK2 protein levels were decreased by 76±6% in cardiac homogenates of homozygous mutant mice but were not changed in other tissues (Figure 2C). GRK5 levels did not change with cardiac GRK2 ablation, showing that there is no compensatory counter-regulation of the other major cardiac-expressed GRK25,26 (Figure 2C). Cardiac structure and echocardiographic ejection performance were normal (Figure 2D), as was myocardial histology (see below). RNA dot-blot analysis showed no change in expression of "fetal" cardiac genes known to be upregulated in cardiac pathology23 (Figure 2E).
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Consistent with our observation that GRK2 protein was not completely eliminated from the myocardial homogenate of cardiac GRK2-del mice, cardiac-specific gene ablation using Cre-lox technology is typically less than 100% effective in the whole heart.27–30 Promoters that drive Cre recombinase expression only in cardiac myocytes leave gene expression intact in nonmyocyte myocardial cells. Also, Cre-mediated gene recombination is subject to kinetic limitations.31 Thus, the potential exists for both intramyocardial chimerism and myocyte-to-myocyte variability in gene recombination. Accordingly, we examined Nkx2.5-Cre–mediated GRK2 ablation in embryonic and adult hearts using PCR. In hearts isolated from E13.5 embryos, qPCR showed 63±3% (n=6) GRK2 recombination, compared with 27% recombination in adult ventricular myocardium (mean of 2) (Figure 3A and 3B). Thus, Nkx2.5-Cre–driven GRK2 ablation was complete (ie, exceeding adult levels) by E13.5. The higher recombination value from embryonic hearts is likely attributable to relative cardiomyocyte enrichment in the embryonic heart, reflecting proportionally fewer nonrecombined nonmyocyte GRK2 alleles in comparison with DNA derived from adult myocardium.
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We also assessed myocyte-to-myocyte variability of Nkx2.5-Cre–mediated GRK2 recombination by comparative PCR analysis of DNA isolated from 36 individual adult ventricular cardiomyocytes. After whole-genome amplification, PCR products were detected in 27 cells, in which complete recombination (absence of any GRK2 loxP allele) was observed in 16, recombination with some residual nonrecombined GRK2 loxP allele in 8 cells, and absence of recombination was observed in 3 cells (Figure 3C). Complete GRK2 deletion in 
60% of adult cardiomyocytes, together with our observation that adult cardiac GRK2del mice are viable, with structurally and functionally normal-appearing hearts, is consistent with the notion that there is no autonomous requirement for cardiomyocyte GRK2 in embryonic heart development after cardiac specification and cardiomyocyte differentiation.
Cardiomyocyte GRK2 Plays a Major Role in Inotropic and Lusitropic Tachyphylaxis to ß-Adrenergic Agonist
The effects of cardiac GRK2 ablation in adult hearts were assessed on its canonical function, desensitization of ß-AR responses.1 In the heart, the most important ß-adrenergic response is increased inotropic activity, typically measured as the maximum rate of change of left ventricular pressure during systole (+dP/dt). Cardiac GRK2del mice exhibited a leftward shift of the peak +dP/dt response to infused doses of isoproterenol (Figure 4A), with an EC50 of 0.4±0.1 ng/g per minute, which was significantly lower that that of 0.7±0.04 ng/g per minute for control GRKf/f mice (n=4, P=0.02). Thus, cardiac GRK2del mice exhibit greater inotropic sensitivity to this nonselective ß-adrenergic agonist. Enhanced inotropic sensitivity to isoproterenol was not the consequence of a GRK2-mediated alteration in ß-AR number because receptor density measured by [125I]CYP binding was the same in cardiac GRK2del hearts (71±6 fmol/mg), compared with GRK2f/f hearts (72±6 fmol/mg) (n=3 each, P=NS, data not shown).
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P=0.057. E, Immunoblot detection of Ser355/356-phosphorylated ß2-AR in hearts from human ß2-AR–overexpressing transgenic mice. C=control, 0 to 20=minutes of 1 µmol/L isoproterenol infusion. Lysate from a nontransgenic heart (ntg) is shown as a negative control. F, Densitometric quantitation of phosphorylated/total ß2-AR during 1 µmol/L isoproterenol infusion shown in E (black squares), with redrawn data from B showing peak positive dP/dt measured during infusion of maximal isoproterenol in wild-type (GRK2f/f) mice (open circles).Tachyphylaxis, the acute agonist-mediated loss of inotropic and lusitropic responsiveness to sustained high-dose isoproterenol, was blunted in cardiac GRK2del mice, as seen by the reduced loss of (ie, maintained) +dP/dt and –dP/dt responses during the isoproterenol infusion (Figure 4B and 4D). In contrast, there were no differences in heart rate responses between mice with and without cardiac GRK2 (Figure 4C).
The mechanism for functional tachyphylaxis is generally accepted to be homologous desensitization of agonist-occupied receptors, for which the specific biochemical events have been defined in detail.1–3 Briefly, GRK-mediated phosphorylation of ligand-bound receptors permits recruitment of ß-arrestin, which uncouples receptors from G-protein effectors, thus terminating signaling and end-organ response despite the continued presence of agonist. To determine whether the time course of inotropic tachyphylaxis during sustained cardiac exposure to isoproterenol correlated with that for GRK-mediated phosphorylation of myocardial ß-ARs, we examined ß-AR phosphorylation in isolated perfused mouse hearts stimulated with 1 µmol/L isoproterenol. Because levels of endogenous cardiac ß-AR are too low for detection of phosphorylation status, and the antibody that specifically recognizes GRK-phosphorylated (S355/356) ß2-ARs is specific for the human receptor, we created a line of mice transgenically expressing human ß2-ARs under control of an attenuated mutant 
-myosin heavy chain promoter,32 such that these mice do not develop the characteristic cardiomyopathy observed with some other ß-AR transgenic models.33 We found that the level of GRK-phosphorylated ß2-AR progressively increased over the 20-minute time period of isoproterenol infusion, whereas total ß-AR immunoreactivity remained constant (Figure 4E). Together with the functional studies, these results show a close temporal association between tachyphylaxis of isoproterenol-stimulated cardiac inotropism and isoproterenol-mediated stimulation of ß-AR phosphorylation by GRKs (Figure 4F). Attenuation of ß-AR tachyphylaxis in cardiac GRK2del mice indicates that GRK2 is a major regulator of ß-adrenergic responsiveness in the heart.
Cardiomyocyte GRK2 Protects Against Catecholamine Cardiomyopathy
Unrestrained ß-AR stimulation, as with forced receptor expression or chronic agonist infusion,33,34 produces myocardial hypertrophy followed by heart failure, designated "catecholamine cardiomyopathy."35 Catecholamine excess is also a hallmark of human heart failure, in which ß-AR blockade has proven to be a life-prolonging therapy.36 Interestingly, one explanation for the benefits of ß-AR blockade in heart failure is that partial antagonism of receptors prevents their agonist-mediated desensitization and downregulation, therefore maintaining ß-AR responsiveness.37 Based on a similar rationale, GRK2 inhibition has shown therapeutic promise in several heart failure models, maintaining cardiac function and ß-AR responsiveness.17,38,39 However, the C-terminal peptide used to inhibit GRK2 also has the potential to interfere broadly with events linked to Gß/
signaling, providing for a different potential mechanism of activation. To clarify the role of GRK2 in catecholamine excess and heart failure, we examined the effect of cardiomyocyte GRK2 deletion on the cardiac response to chronic, systemic infusion of isoproterenol.
Preliminary dose-ranging experiments showed that control mice tolerated 15 and 30 mg/kg per day, but not 60 mg/kg per day, isoproterenol infusion for 14 days, during which time echocardiographic left ventricular mass significantly increased (Figure 5A), consistent with the findings of previous studies.34 Ventricular contractility (measured as Vcfc) was not significantly altered during the 2-week administration of 15 or 30 mg/kg per day isoproterenol, but declined precipitously in mice treated with 60 mg/kg per day isoproterenol, in which the study had to be terminated after 8 days (Figure 5B). Because our acute studies had shown that cardiomyocyte GRK2 deletion increased cardiac sensitivity to isoproterenol and delayed ß-AR desensitization (vide supra), we performed comparative studies with the largest dose of isoproterenol that was well tolerated, 30 mg/kg per day. After isoproterenol for 14 days, both control GRK2f/f and cardiac GRK2del mice developed cardiac dilation and increased left ventricular mass (Figure 5C and the Table in the online data supplement, available at http://circres.ahajournals.org). However, only GRK2del mouse hearts showed adverse remodeling (decreased h/r, wall thickness/chamber radius ratio) and decreased contractile performance, measured as Vcfc (Figure 5D) or fractional shortening (supplemental Table). The histological picture of chronic isoproterenol-treated GRK2del mouse hearts was also more severe, with evidence of generalized interstitial and replacement fibrosis (Figure 5E). Thus, absence of GRK2 in cardiac myocytes exacerbates myocardial toxicity of catecholamine excess and contributes to cardiomyopathy development and functional decompensation.
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Discussion |
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Two critical functions have been postulated for GRK2 in the heart: enabling embryonic cardiac development through an unidentified mechanism11; and regulating cardiac contractile function through modulation of ß-adrenergic coupling to G-protein signaling effectors.12 Here, we have examined each of these roles through the use of conditional gene ablation.
Germline deletion of GRK2 using EIIa-directed Cre, which is specifically expressed in the preimplantation embryo,20 resulted in embryonic lethality at approximately E10.5. At E9.5, there was evidence of growth retardation and delayed heart development in the GRK2 null embryos. Cardiac looping and specification appeared normal, but the presence of pericardial edema in some embryos is consistent with cardiac failure. Although these findings are in general agreement with the previously described phenotype of "thin myocardium syndrome" and depressed ventricular ejection fraction with embryonic lethality at E15.5 after conventional GRK2 ablation,11 there are intriguing differences between the current and previous studies. Here, embryonic growth retardation and lethality were observed much earlier in development. We found that cardiac development did not progress beyond the looping stage, and although the myocardium was only a single cell thick, expression of Nkx2.5 showed that cardiomyocyte specification and differentiation had occurred normally. In the previous study, cardiac development proceeded to the 4-chambered heart stage, but wall thickness and ventricular trabeculation were abnormal. The reason for these differences is not known and may involve different knockout strategies or differences in mouse genetic background. Although embryonic lethality and suggestions of heart failure in both models seem to implicate GRK2 involvement in cardiac development, we believe that caution is warranted in this interpretation, as growth retardation and signs of cardiac failure can be the consequence either of a primary cardiac abnormality such as ablation of Nkx2.5,40 or of noncardiac or generalized defects in embryonic development. For example, abnormal formation of the vasculature can produce phenotypes that mimic impaired heart development.24
To determine whether GRK2 was required for cardiac development, we used one of the earliest genetic determinants of myocardial differentiation, Nkx2.5, to drive Cre-mediated GRK2 ablation in the early embryonic heart. Based on the time course of recombination achieved by the Nkx2.5-Cre knock-in in ROSA26 Cre indicator mice,21 we anticipated that cardiac GRK2 ablation would occur early during embryonic heart development. Indeed, GRK2 gene recombination, although less than 100% effective, was complete (exceeding adult levels) by E13.5. Studies of isolated adult cardiomyocytes showed that recombination of all 4 alleles (2 alleles per nucleus in a binucleated cell) occurred in approximately two-thirds of cells. Thus, cardiomyocyte differentiation, proliferation, and incorporation into myocardium does not require cell-autonomous expression of GRK2.
One possible way of resolving the apparently disparate findings of generalized embryonic growth retardation, delayed cardiac development, and early lethality with germline GRK2 ablation, but viable and outwardly normal mice with embryonic cardiomyocyte-specific GRK2 ablation, is that there is a critical extracardiac function specific for GRK2 in embryonic development. For example, GRK2 has the potential to modify the signaling activity of Smoothened (Smo), a 7-transmembrane spanning protein without a ligand that transduces Hedgehog signaling.41 The essential role of Smo in embryonic development has been established through ablation of the Smo gene,41 and Smo was recently shown to be a substrate for GRK2-mediated phosphorylation.42–44 However, whether GRK2-mediated phosphorylation of Smo affects its signal transduction during embryonic development is not currently known.
Adult mice largely lacking cardiomyocyte GRK2 provided an opportunity to define the role of GRK2 on the acute and chronic cardiac responses to ß-AR stimulation. Cardiomyocyte-specific deletion of GRK2 significantly enhanced contractile responsiveness to acutely infused ß-adrenergic agonist, consistent with previous studies using GRK2 inhibition.12 We also demonstrated that acute tachyphylaxis to isoproterenol in cardiac GRK2del mice was strikingly diminished, compared with wild-type GRK2f/f mice. These results define a major contribution of GRK2 to cardiac ß-adrenergic tachyphylaxis, and by inference, to homologous desensitization of ß-ARs. The less-than-complete elimination of tachyphylaxis may reflect, in part, the less-than-totally effective ablation of GRK2 in cardiomyocytes using Nkx2.5 Cre-lox recombination, although it is likely that alternate desensitization pathways mediated by GRK5, which is also highly expressed in myocardium and can desensitize ß-ARs,25,26 or protein kinase A,45 can also modulate cardiac ß-adrenergic responsiveness.
Regulation of contractility represents the acute "fight or flight" function of ß-adrenergic signaling, but there is increasing evidence that chronic ß-adrenergic stimulation also impacts ventricular function by stimulating cardiomyocyte hypertrophy and by causing cardiomyocyte toxicity.33,46 Indeed, inhibition of GRK2 with the ß-ARKct inhibitory peptide has improved the outcome of several forms of experimental heart failure.17,38,39 However, our studies suggest that absence of GRK2, with the resulting lack of restraint on ß-AR signaling, accelerates the cardiomyopathy that develops under conditions of chronic catecholamine stimulation. These findings suggest that additional or alternate mechanisms may be operative in the therapeutic effects of ß-ARKct and that GRK2 inhibition may need to be carefully titrated to avoid undesirable consequences.
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Acknowledgments |
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Sources of Funding
Supported by National Heart, Lung, and Blood Institute grants HL59888, HL58010, HL77101, and HL69779.
Disclosures
None.
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Footnotes |
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Original received May 16, 2006; revision received September 14, 2006; accepted September 18, 2006.
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References |
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1. Kohout TA, Lefkowitz RJ. Regulation of G protein-coupled receptor kinases and arrestins during receptor desensitization. Mol Pharmacol. 2003; 63: 9–18.
2. Fredriksson R, Lagerstrom MC, Lundin LG, Schioth HB. The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol Pharmacol. 2003; 63: 1256–1272.
3. Luttrell LM, Lefkowitz RJ. The role of beta-arrestins in the termination and transduction of G-protein-coupled receptor signals. J Cell Sci. 2002; 115: 455–465.
4. Lefkowitz RJ, Shenoy SK. Transduction of receptor signals by beta-arrestins. Science. 2005; 308: 512–517.
5. Lyubarsky AL, Chen C, Simon MI, Pugh EN Jr. Mice lacking G-protein receptor kinase 1 have profoundly slowed recovery of cone-driven retinal responses. J Neurosci. 2000; 20: 2209–2217.
6. Peppel K, Boekhoff I, McDonald P, Breer H, Caron MG, Lefkowitz RJ. G protein-coupled receptor kinase 3 (GRK3) gene disruption leads to loss of odorant receptor desensitization. J Biol Chem. 1997; 272: 25425–25428.
7. Walker JK, Gainetdinov RR, Feldman DS, McFawn PK, Caron MG, Lefkowitz RJ, Premont RT, Fisher JT. G protein-coupled receptor kinase 5 regulates airway responses induced by muscarinic receptor activation. Am J Physiol Lung Cell Mol Physiol. 2004; 286: L312–L319.
8. Gainetdinov RR, Bohn LM, Walker JK, Laporte SA, Macrae AD, Caron MG, Lefkowitz RJ, Premont RT. Muscarinic supersensitivity and impaired receptor desensitization in G protein-coupled receptor kinase 5-deficient mice. Neuron. 1999; 24: 1029–1036.[CrossRef][Medline] [Order article via Infotrieve]
9. Gainetdinov RR, Bohn LM, Sotnikova TD, Cyr M, Laakso A, Macrae AD, Torres GE, Kim KM, Lefkowitz RJ, Caron MG, Premont RT. Dopaminergic supersensitivity in G protein-coupled receptor kinase 6-deficient mice. Neuron. 2003; 38: 291–303.[CrossRef][Medline] [Order article via Infotrieve]
10. Benovic JL, Strasser RH, Caron MG, Lefkowitz RJ. Beta-adrenergic receptor kinase: identification of a novel protein kinase that phosphorylates the agonist-occupied form of the receptor. Proc Natl Acad Sci U S A. 1986; 83: 2797–2801.
11. Jaber M, Koch WJ, Rockman H, Smith B, Bond RA, Sulik KK, Ross J Jr, Lefkowitz RJ, Caron MG, Giros B. Essential role of beta-adrenergic receptor kinase 1 in cardiac development and function. Proc Natl Acad Sci U S A. 1996; 93: 12974–12979.
12. Koch WJ, Rockman HA, Samama P, Hamilton RA, Bond RA, Milano CA, Lefkowitz RJ. Cardiac function in mice overexpressing the ß-adrenergic receptor kinase or a ßARK inhibitor. Science. 1995; 268: 1350–1353.
13. Bristow MR, Hershberger RE, Port JD, Gilbert EM, Sandoval A, Rasmussen R, Cates AE, Feldman AM. Beta-adrenergic pathways in nonfailing and failing human ventricular myocardium. Circulation. 1990; 82 (suppl I): I-12–I-25.[Medline] [Order article via Infotrieve]
14. Gros R, Benovic JL, Tan CM, Feldman RD. G-protein-coupled receptor kinase activity is increased in hypertension. J Clin Invest. 1997; 99: 2087–2093.[Medline] [Order article via Infotrieve]
15. Ungerer M, Kessebohm K, Kronsbein K, Lohse MJ, Richardt G. Activation of beta-adrenergic receptor kinase during myocardial ischemia. Circ Res. 1996; 79: 455–460.
16. Manning BS, Shotwell K, Mao L, Rockman HA, Koch WJ. Physiological induction of a beta-adrenergic receptor kinase inhibitor transgene preserves beta-adrenergic responsiveness in pressure-overload cardiac hypertrophy. Circulation. 2000; 102: 2751–2757.
17. Rockman HA, Chien KR, Choi DJ, Iaccarino G, Hunter JJ, Ross J Jr, Lefkowitz RJ, Koch WJ. Expression of a beta-adrenergic receptor kinase 1 inhibitor prevents the development of myocardial failure in gene-targeted mice. Proc Natl Acad Sci U S A. 1998; 95: 7000–7005.
18. White DC, Hata JA, Shah AS, Glower DD, Lefkowitz RJ, Koch WJ. Preservation of myocardial beta-adrenergic receptor signaling delays the development of heart failure after myocardial infarction. Proc Natl Acad Sci U S A. 2000; 97: 5428–5433.
19. Farley FW, Soriano P, Steffen LS, Dymecki SM. Widespread recombinase expression using FLPeR (flipper) mice. Genesis. 2000; 28: 106–110.[CrossRef][Medline] [Order article via Infotrieve]
20. Dooley TP, Miranda M, Jones NC, DePamphilis ML. Transactivation of the adenovirus EIIa promoter in the absence of adenovirus E1A protein is restricted to mouse oocytes and preimplantation embryos. Development. 1989; 107: 945–956.
21. Moses KA, DeMayo F, Braun RM, Reecy JL, Schwartz RJ. Embryonic expression of an Nkx2–5/Cre gene using ROSA26 reporter mice. Genesis. 2001; 31: 176–180.[CrossRef][Medline] [Order article via Infotrieve]
22. Liberatore CM, Searcy-Schrick RD, Vincent EB, Yutzey KE. Nkx-2.5 gene induction in mice is mediated by a Smad consensus regulatory region. Dev Biol. 2002; 244: 243–256.[CrossRef][Medline] [Order article via Infotrieve]
23. D’Angelo DD, Sakata Y, Lorenz JN, Boivin GP, Walsh RA, Liggett SB, Dorn GW. Transgenic Galphaq overexpression induces cardiac contractile failure in mice. Proc Natl Acad Sci U S A. 1997; 94: 8121–8126.
24. Conway SJ, Kruzynska-Frejtag A, Kneer PL, Machnicki M, Koushik SV. What cardiovascular defect does my prenatal mouse mutant have, and why? Genesis. 2003; 35: 1–21.[CrossRef][Medline] [Order article via Infotrieve]
25. Kunapuli P, Benovic JL. Cloning and expression of GRK5: a member of the G protein-coupled receptor kinase family. Proc Natl Acad Sci U S A. 1993; 90: 5588–5592.
26. Premont RT, Koch WJ, Inglese J, Lefkowitz RJ. Identification, purification, and characterization of GRK5, a member of the family of G protein-coupled receptor kinases. J Biol Chem. 1994; 269: 6832–6841.
27. Agah R, Frenkel PA, French BA, Michael LH, Overbeek PA, Schneider MD. Gene recombination in postmitotic cells. Targeted expression of Cre recombinase provokes cardiac-restricted, site-specific rearrangement in adult ventricular muscle in vivo. J Clin Invest. 1997; 100: 169–179.[Medline] [Order article via Infotrieve]
28. Shai SY, Harpf AE, Babbitt CJ, Jordan MC, Fishbein MC, Chen J, Omura M, Leil TA, Becker KD, Jiang M, Smith DJ, Cherry SR, Loftus JC, Ross RS. Cardiac myocyte-specific excision of the beta1 integrin gene results in myocardial fibrosis and cardiac failure. Circ Res. 2002; 90: 458–464.
29. Jacoby JJ, Kalinowski A, Liu MG, Zhang SS, Gao Q, Chai GX, Ji L, Iwamoto Y, Li E, Schneider M, Russell KS, Fu XY. Cardiomyocyte-restricted knockout of STAT3 results in higher sensitivity to inflammation, cardiac fibrosis, and heart failure with advanced age. Proc Natl Acad Sci U S A. 2003; 100: 12929–12934.
30. Henderson SA, Goldhaber JI, So JM, Han T, Motter C, Ngo A, Chantawansri C, Ritter MR, Friedlander M, Nicoll DA, Frank JS, Jordan MC, Roos KP, Ross RS, Philipson KD. Functional adult myocardium in the absence of Na+-Ca2+ exchange: cardiac-specific knockout of NCX1. Circ Res. 2004; 95: 604–611.
31. Ringrose L, Lounnas V, Ehrlich L, Buchholz F, Wade R, Stewart AF. Comparative kinetic analysis of FLP and Cre recombinases: mathematical models for DNA binding and recombination. J Mol Biol. 1998; 284: 363–384.[CrossRef][Medline] [Order article via Infotrieve]
32. Odley A, Hahn HS, Lynch RA, Marreez Y, Osinska H, Robbins J, Dorn GW. Regulation of cardiac contractility by Rab4-modulated beta2-adrenergic receptor recycling. Proc Natl Acad Sci U S A. 2004; 101: 7082–7087.
33. Liggett SB, Tepe NM, Lorenz JN, Canning AM, Jantz TD, Mitarai S, Yatani A, Dorn GW. Early and delayed consequences of beta(2)-adrenergic receptor overexpression in mouse hearts: critical role for expression level. Circulation. 2000; 101: 1707–1714.
34. Kudej RK, Iwase M, Uechi M, Vatner DE, Oka N, Ishikawa Y, Shannon RP, Bishop SP, Vatner SF. Effects of chronic beta-adrenergic receptor stimulation in mice. J Mol Cell Cardiol. 1997; 29: 2735–2746.[CrossRef][Medline] [Order article via Infotrieve]
35. Dorn GW. Adrenergic pathways and left ventricular remodeling. J Card Fail. 2002; 8: S370–S373.[CrossRef][Medline] [Order article via Infotrieve]
36. Packer M. Current role of beta-adrenergic blockers in the management of chronic heart failure. Am J Med. 2001; 110 (suppl 7A): 81S–94S.[Medline] [Order article via Infotrieve]
37. Bristow MR. Beta-Adrenergic receptor blockade in chronic heart failure. Circulation. 2000; 101: 558–569.
38. Rockman HA, Choi D-J, Akhter SA, Jaber M, Giros B, Lefkowitz RJ, Caron MG, Koch WJ. Control of myocardial contractile function by the level of ß-adrenergic receptor kinase 1 in gene-targeted mice. J Biol Chem. 1998; 273: 18180–18184.
39. Tachibana H, Naga Prasad SV, Lefkowitz RJ, Koch WJ, Rockman HA. Level of ß-adrenergic receptor kinase 1 inhibition determines degree of cardiac dysfunction after chronic pressure overload-induced heart failure. Circulation. 2005; 111: 591–597.
40. Tanaka M, Chen Z, Bartunkova S, Yamasaki N, Izumo S. The cardiac homeobox gene Csx/Nkx2.5 lies genetically upstream of multiple genes essential for heart development. Development. 1999; 126: 1269–1280.[Abstract]
41. Zhang XM, Ramalho-Santos M, McMahon AP. Smoothened mutants reveal redundant roles for Shh and Ihh signaling including regulation of L/R asymmetry by the mouse node. Cell. 2001; 105: 781–792.[CrossRef][Medline] [Order article via Infotrieve]
42. Chen W, Ren XR, Nelson CD, Barak LS, Chen JK, Beachy PA, de Sauvage F, Lefkowitz RJ. Activity-dependent internalization of smoothened mediated by beta-arrestin 2 and GRK2. Science. 2004; 306: 2257–2260.
43. Wilbanks AM, Fralish GB, Kirby ML, Barak LS, Li YX, Caron MG. Beta-arrestin 2 regulates zebrafish development through the hedgehog signaling pathway. Science. 2004; 306: 2264–2267.
44. Meloni AR, Fralish GB, Kelly P, Salahpour A, Chen JK, Wechsler-Reya RJ, Lefkowitz RJ, Caron MG. Smoothened signal transduction is promoted by G protein coupled receptor kinase 2. Mol Cell Biol. 2006; 26: 7550–7560.
45. Lefkowitz RJ, Hausdorff WP, Caron MG. Role of phosphorylation in desensitization of the beta-adrenoceptor. Trends Pharmacol Sci. 1990; 11: 190–194.[CrossRef][Medline] [Order article via Infotrieve]
46. Karoor V, Vatner SF, Takagi G, Yang G, Thaisz J, Sadoshima J, Vatner DE. Propranolol prevents enhanced stress signaling in Gs alpha cardiomyopathy: potential mechanism for beta-blockade in heart failure. J Mol Cell Cardiol. 2004; 36: 305–312.[CrossRef][Medline] [Order article via Infotrieve]
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