G Protein–Coupled Receptor Kinase 2 Ablation in Cardiac Myocytes Before or After Myocardial Infarction Prevents Heart Failure
Myocardial G protein–coupled receptor kinase (GRK)2 is a critical regulator of cardiac β-adrenergic receptor (βAR) signaling and cardiac function. Its upregulation in heart failure may further depress cardiac function and contribute to mortality in this syndrome. Preventing GRK2 translocation to activated βAR with a GRK2-derived peptide that binds Gβγ (βARKct) has benefited some models of heart failure, but the precise mechanism is uncertain, because GRK2 is still present and βARKct has other potential effects. We generated mice in which cardiac myocyte GRK2 expression was normal during embryonic development but was ablated after birth (αMHC-Cre×GRK2 fl/fl) or only after administration of tamoxifen (αMHC-MerCreMer×GRK2 fl/fl) and examined the consequences of GRK2 ablation before and after surgical coronary artery ligation on cardiac adaptation after myocardial infarction. Absence of GRK2 before coronary artery ligation prevented maladaptive postinfarction remodeling and preserved βAR responsiveness. Strikingly, GRK2 ablation initiated 10 days after infarction increased survival, enhanced cardiac contractile performance, and halted ventricular remodeling. These results demonstrate a specific causal role for GRK2 in postinfarction cardiac remodeling and heart failure and support therapeutic approaches of targeting GRK2 or restoring βAR signaling by other means to improve outcomes in heart failure.
The role of myocardial β-adrenergic receptor (βAR) signaling in heart failure (HF) is controversial, with abundant data supporting dominant effects that are either pathological or beneficial. Pathological effects are strongly suggested by the cardiomyopathies that develop under conditions of chronic βAR activation after long-term catecholamine administration1 or as a consequence of high levels of overexpressed cardiac β1- or β2-ARs.2,3 Likewise, enhanced signaling from overexpression of the βAR signal transducer Gαs causes HF.4 Most importantly, however, are the human patient data indicating that βAR signaling is deleterious in HF: genetic polymorphisms that enhance myocardial sympathetic tone are independent risk factors for HF,5 and pharmacological βAR antagonism has been known for more than 2 decades to strikingly prolong life when used as primary therapy in HF.6
Despite the strength of data in experimental systems and the human condition that excessive βAR signaling can contribute to HF, there continue to be those who advocate modulating βARs in HF as a form of therapy.7 This viewpoint is based on observations that 2 transgenic mouse lines overexpressing low levels of β2-AR in the heart, which were developed in 2 independent laboratories, demonstrated enhanced contractile function without myocardial disease,8,9 and β2-ARs can improve function of cardiomyopathy models.10,11 Another group has suggested that transgenic or adenoviral-mediated overexpression of the downstream βAR signaling effector adenylyl cyclase has similar effects.12 Also consistent with this idea are studies pioneered in our laboratory in which prevention of typical HF-associated G protein–coupled receptor kinase (GRK)2-mediated desensitization and downregulation of myocardial βARs has rescued numerous genetic13,14 and physiological15,16 models of HF.
The reasons for the apparently contradictory datasets relating to βAR signaling in failing myocardium are unknown, but understanding them will be essential to fully optimize HF treatment. Regarding GRK2 inhibition, specifically, concerns have been expressed about the specificity and true mechanism of action of the βARKct GRK2 inhibitor used in many of these studies.17 It is also not entirely clear that GRK2 is the best target in HF, because GRK3, GRK5, and GRK6 are also expressed in the heart, and forced expression of GRK5 had similar desensitization effects on myocardial βARs as does forced expression of GRK2.18 Indeed, cardiac-specific ablation of GRK2 in the early embryo actually sensitized adult mice to catecholamine cardiomyopathy, rather than conferring protection, which was interpreted as evidence for pathological effects of βAR signaling.19 These results raise additional questions about whether GRK2 inhibition may have different effects in different experimental HF models or at different time points relative to the induction of HF.
To address these issues, we created mice in which the GRK2 gene could be selectively ablated in cardiac myocytes in a temporally defined manner and compared the consequences of heart-specific GRK2 ablation before and after induction of HF by myocardial infarction (MI).
Materials and Methods
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Conditional mice bearing floxed GRK2 alleles (GRK2 fl/fl) have previously been described.19 Transgenic mice overexpressing Cre-recombinase protein fused to 2 mutant estrogen receptor ligand-binding domains under the control of the α-myosin heavy chain (αMHC) promoter (αMHC-MerCreMer)20 were received from The Jackson Laboratory (JAX Mice and Services, Bar Harbor, Me). Homozygous mice with the floxed GRK2 alleles were crossed with αMHC-MerCreMer mice, and the resulting αMHC-MerCreMer×GRK2 fl/fl mice and GRK2 fl/fl, as well as αMHC-MerCreMer, were studied. To induce Cre recombination and subsequent deletion of GRK2 adult mice were treated with tamoxifen (Tmx) (Sigma-Aldrich, St Louis, Mo).20
In addition, αMHC-Cre mice21 were bred on to the GRK2 fl/fl background to generate cardiac GRK2-deleted mice, initiated by the activation of the αMHC-promoter. All animal procedures and experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of Thomas Jefferson University.
Model of Left Ventricular MI
MI was induced by high ligation of the left anterior descending coronary artery, as described previously.22
Echocardiographic and Hemodynamic Analysis of Cardiac Function
Left ventricular (LV) function was assessed by transthoracic echocardiography. LV hemodynamics were determined in anesthetized mice under baseline conditions and stimulation with the unselective βAR agonist isoproterenol (Sigma-Aldrich)23 and after a single dose of the selective β2-AR agonist fenoterol (Sigma-Aldrich). The peak response, which typically occurred 2 minutes after the injection of fenoterol, was used as readout for the intensity of the β2-AR signal.
Isolation of Cardiac Myocytes
Adult mouse cardiac myocytes were isolated from sham and infarcted GRK2 fl/fl and αMHC-Cre×GRK2 fl/fl mice, as previously described.24
Single Myocyte Contractility Studies
Isolated cardiac myocytes were stimulated in an electric field, and continuously flushed with tyrodes containing 1 mmol/L CaCl2 without (baseline) and with 10−8 mol/L isoproterenol (isoproterenol-stimulation). Single-cell contractions were measured by video edge detection (Fluorescence and Contractility System, IonOptix, Milton, Mass).
RNA Isolation, Reverse Transcription, and Quantitative Real-Time RT-PCR
Isolated LV cardiac myocytes (rescue study) or myocardial tissue from the remote zone (prevention-study) was snap-frozen. Quantitative real-time polymerase chain reaction was performed as described previously.22
Western Blot Analysis
Western blotting was performed as described previously.22 Cardiac protein levels of GRK2 (sc-562, C-15, Santa Cruz Biotechnology, 1:5000), α-actin (A7811, Sigma-Aldrich, 1:5000), and calsequestrin (CSQ) (208915; Calbiochem; 1:10 000) were assessed in cardiac myocyte cellular preparations.
Data are generally expressed as means±SEM. An unpaired 2-tailed t test, a 1-way ANOVA, and a 2-way repeated measurement ANOVA were performed for between-group comparisons. Survival analysis was performed by the Kaplan–Meier method, and between-group differences in survival were tested by the log-rank test. For all tests, a probability value of <0.05 was considered significant.
Defined Loss of GRK2 in Cardiac Myocytes After MI Rescues the HF Phenotype
Survival of HF Is Improved After Loss of GRK2
To gain insight into whether GRK2 ablation could be therapeutic in hearts already in HF, we used Tmx-inducible GRK2 knockout (KO) (αMHC-MerCreMer×GRK2 fl/fl) mice. These mice along with αMHC-MerCreMer mice as controls were subjected to coronary artery ligation and loss of GRK2 was induced by 5 consecutive days of Tmx injections (on days 10 to 14 post-MI) (Figure 1A and 1B). We found similar mortality in both mice until ≈12 to 13 days post-MI (Figure 1C). Interestingly, following the Tmx injections, survival in Tmx-treated αMHC-MerCreMer×GRK2 fl/fl mice was significantly preserved compared to the continued death seen in Tmx-treated control mice (Figure 1C). Thus, the loss of cardiac GRK2 after MI prevents the significant mortality seen chronically in HF mice with normal levels of GRK2 in their hearts.
Loss of Cardiac GRK2 in Already Established HF Rescues Cardiac Function
Importantly, all groups of mice 10 days post-MI had similar levels of chamber dilatation and cardiac dysfunction compared to pre-MI measurements, as assessed by echocardiography (Figure 2A and 2B). The 3 sham groups showed no alterations in cardiac function or dimensions at any time point. Following loss of GRK2 in αMHC-MerCreMer×GRK2 fl/fl mice, echocardiography at 42 and 120 days post-MI showed stabilization of LV remodeling (Figure 2A). Furthermore, significant enhancement of cardiac function (as measured by fractional shortening percentage [FS]) was observed after the loss of cardiac GRK2 compared to both groups of Tmx-treated control mice (αMHC-MerCreMer and GRK2 fl/fl) (Figure 2B).
Measurements of LV hemodynamics 120 days post-MI showed preserved responsiveness to βAR stimulation in infarcted Tmx-treated αMHC-MerCreMer×GRK2 fl/fl mice, whereas infarcted Tmx-treated αMHC-MerCreMer mice revealed an impaired βAR reserve compared to sham mice (Figure 2C and 2D). LV end-diastolic pressure (LVEDP) was significantly lower in infarcted αMHC-MerCreMer×GRK2 fl/fl mice as compared to infarcted αMHC-MerCreMer mice (Figure 2E).
Loss of GRK2 in Post-MI Failing Cardiac Myocytes Reduces Activation of the Fetal Gene Program
RT-PCR analysis was performed 5 weeks post-MI (3 weeks after the last dose of Tmx) on cardiac myocytes isolated from the LV. Interestingly, less induction of selected fetal genes (atrial natriuretic peptide [ANP], brain natriuretic peptide [BNP], and βMHC) was found in infarcted Tmx-treated αMHC-MerCreMer×GRK2 fl/fl mice as compared to post-MI Tmx-treated αMHC-MerCreMer cardiac myocytes (Figure 3).
We also examined expression of GRK mRNA post-MI in cardiac myocytes isolated from the LV. As expected in control Tmx-treated αMHC-MerCreMer mice, MI produced a significant increase in GRK2 expression over sham levels, whereas infarcted Tmx-treated αMHC-MerCreMer×GRK2 fl/fl mice showed significantly lower GRK2 levels (Figure 3D). Interestingly, GRK5 mRNA was selectively upregulated in infarcted Tmx-treated αMHC-MerCreMer mice (Figure V in the online data supplement).
Loss of GRK2 Before MI Prevents the Development of HF
Loss of Cardiac Myocyte GRK2 Before MI Reduces Infarct-Related Mortality
To study the consequence of GRK2 loss on the development of HF, adult (8-week-old) αMHC-Cre×GRK2 fl/fl mice and their corresponding littermate controls (GRK2 fl/fl) were subjected to coronary artery ligation and subsequent MI (Figure 4A). αMHC-Cre×GRK2 fl/fl mice have an ≈80% loss of cardiac myocyte GRK2 (Figure 4B). Of note, infarct size was not different 24 hours or 28 days after MI between mice with normal levels of GRK2 and the GRK2 KO mice (supplemental Figure II). Interestingly, survival was significantly improved in male αMHC-Cre×GRK2 fl/fl mice subjected to MI compared to control GRK2 fl/fl mice (see Figure 4C). There was a trend toward improved survival in infarcted αMHC-Cre×GRK2 fl/fl mice of both sexes (P=0.06) (supplemental Figure III). The differences in survival were present, despite only the αMHC-Cre×GRK2 fl/fl mice and not the control mice in this study (GRK2 fl/fl) possessed Cre recombinase, which has been proven previously to contribute to cardiac pathology.25
Loss of GRK2 in Cardiac Myocytes Before MI Reduces the Extent and Progression of Cardiac Dysfunction
In vivo cardiac function was assessed by echocardiography before and 28 days after MI (or sham). Twenty-eight days after MI, control GRK2 fl/fl mice displayed significantly depressed systolic function and enlarged cardiac chambers as compared to pre-MI values (Figure 5A and 5B). In striking contrast, αMHC-Cre×GRK2 fl/fl mice subjected to MI did not display the same degree of cardiac deterioration (Figure 5A and B).
Loss of Myocyte GRK2 Before MI Improves LV Hemodynamics and Preserves βAR Responsiveness
LV contractility (as measured by dP/dtmax) and LV relaxation (as measured by LV dP/dtmin) after isoproterenol were significantly impaired in control GRK2 fl/fl mice compared to sham control mice, demonstrating a loss of inotropic reserve consistent with HF (Figure 5C and 5D). Similarly, hemodynamic studies on αMHC-Cre×GRK2 fl/fl mice revealed signs of cardiac dysfunction at baseline; however, responses to isoproterenol were significantly improved compared to post-MI control mice showing improved contractile reserve and preserved βAR signaling in hearts without GRK2 (Figure 5C and 5D). Interestingly, the response to fenoterol was significantly enhanced in αMHC-Cre×GRK2 fl/fl mice post-MI compared to GRK2 fl/fl mice, arguing in favor of facilitated β2-AR signaling with GRK2 silencing (Figure 5E and 5F). Finally, elevated LVEDP in control post-MI GRK2 fl/fl mice was significantly lowered in mice with GRK2 expression lost (Figure 5G).
Loss of GRK2 Is Associated With a Lower Extent of Cardiac Hypertrophy and Normalized Gene Expression After MI
Heart-to-body weight (HW/BW) ratio was significantly increased 28 days post-MI in control GRK2 fl/fl mice compared to corresponding sham-operated animals (Figure 5H). Although αMHC-Cre×GRK2 fl/fl mice subjected to MI displayed LV hypertrophy compared to sham-operated controls, the extent of hypertrophy was significantly less than that observed in GRK2 fl/fl mice subjected to MI (Figure 5H).
Consistent with decreased hypertrophy in GRK2 KO mice, RT-PCR analysis of the remote (nonischemic) portion of the LV 28 days post-MI showed significantly less induction of selected fetal genes (ANP, BNP, and βMHC) as seen in post-MI control hearts, which showed significant upregulation of these genes compared to sham-controls (Figure 6A through 6C). Upregulation of the mRNAs encoding matrix metalloproteinase-9 and collagen-1 (Coll-1) in the nonischemic LV zone were also seen in post-MI GRK2 fl/fl mice, indicating adverse remodeling; however, in GRK2 KO mice, these changes in matrix metalloproteinase-9 and collagen-1 were not seen (Figure 6D and 6E). These observations in combination with the attenuation of LV chamber dilation (see above) argue in favor of significant repression of adverse LV remodeling by the loss of GRK2 in myocytes.
Furthermore, MI in control mice led to a significant increase in GRK2 mRNA expression over sham levels, which was absent in GRK2 KO mice (Figure 6F). Interestingly, GRK5, another major GRK found in the heart,26 which was upregulated in post-MI control (GRK2 fl/fl) hearts, was not significantly changed in the hearts of post-MI αMHC-Cre×GRK2 fl/fl mice compared to sham (Figure 6G). The fact that the GRK2 KO mice had no increase in GRK5 argues for a lack of compensation by this GRK. Of note, GRK3 a close homologue of GRK2 was not altered in any of our groups post-MI (data not shown).
Loss of GRK2 Normalizes βAR Responsiveness of Single Cardiac Myocytes Post-MI
Figure 7A shows representative steady-state twitches from cardiac myocytes (from sham and infarcted GRK2 fl/fl and αMHC-Cre×GRK2 fl/fl mice) under baseline conditions (left) and following isoproterenol stimulation (10−8 mol/L) (right). Isolated cardiac myocytes from post-MI αMHC-Cre×GRK2 fl/fl mice demonstrated significantly enhanced FS, improved rate of cell shortening (+dL/dt) and improved rate of relengthening (−dL/dt) under isoproterenol stimulation compared to control, post-MI GRK2(fl/fl) myocytes (Figure 7B and 7C). These results indicate that βAR responsiveness post-MI is preserved by the loss of GRK2.
The incidence of HF is increasing, and its long-term prognosis has remained at 50% survival after 5 years despite the broad application of life-saving antineurohormonal pharmacological therapies with angiotensin-converting enzyme inhibition and β-blockade more than 2 decades ago.6 Thus, more efficient HF therapeutics are needed, which may be achieved by identifying nontraditional molecular targets downstream of the neurohormonal ligand-receptor interaction. For a number of years, we have proposed that GRK2, a kinase that regulates the signaling activity and number of βAR in the heart and that, itself, is regulated in HF, might represent such a nontraditional target.27 Indeed, transgenic and adenoviral expression of a peptide inhibitor of GRK2 translocation, βARKct, has benefited a number of experimental HF models.13,14 Mechanistically, however, questions have arisen about the specific effects of βARKct and about the specific pathophysiological role of GRK2 in heart disease. In the present studies, we attempted to unambiguously define the role of GRK2 in postischemic HF and to assess the efficacy of its genetic ablation in a highly relevant model of HF, MI, and at clinically relevant times in the course of the disease, ie, both before and after the inciting ischemic event. In short, our data contains definitive evidence that GRK2 contributes to ischemic HF, and demonstrate a critical role for this kinase in the disease. Moreover our data show the striking efficacy of restoring cardiac βAR signaling homeostasis by GRK2 ablation either before or after MI.
GRK2 Is the Critical GRK in the Failing Heart
Using constitutive (starting postnatally) αMHC-Cre or inducible αMHC-MerCreMer mice, we were able to test the role of GRK2 expression in adult cardiac myocytes in both the development of and rescue of ischemic cardiomyopathy. The use of these mice is necessary because global GRK2 KO mice die in utero.19 Interestingly, mortality post-MI was similar between groups until the Tmx treatment when the induced loss of GRK2 prevented further death up to 120 days post-MI. Thus, the loss of GRK2 expression in myocytes after MI, when HF was already established, clearly led to significantly improved survival chronically after MI. Furthermore, loss of GRK2 before MI significantly reduced mortality in male GRK2 KO mice. In addition, GRK2 abolishment prevented LV dilatation and deterioration of cardiac function that is seen in control mice post-MI. This is the first study showing that controlled loss of GRK2 in failing cardiac myocytes rescues a model of established HF and directly demonstrates that minimizing GRK2 activity in the failing heart is beneficial. Although the loss of GRK2 was not complete, this loss of HF-associated upregulation of cardiac myocyte GRK2 essentially restored in vivo cardiac systolic and diastolic function. Our data strongly suggest GRK2 as the primary GRK critically involved in the functional regulation of the failing heart.
Restoration of βAR Signaling in the Failing Heart by GRK2 Ablation Similar to Effects of the GRK2 Inhibitor Peptide βARKct
Both early and late following the acute inciting event, our present data provide further support for the benefits of restoration or normalization of βAR signaling in HF. The increased LV hemodynamic response and the improved contractility of isolated single LV cardiac myocytes to βAR stimulation in mice lacking cardiac myocyte GRK2 show that signaling through this inotropic system is preserved despite the presence of a large MI. Moreover, these data are consistent with previous rescue studies using the βARKct and demonstrate and support the primary mechanism of action of this Gβγ-sequestering peptide in HF therapeutics to be GRK2 inhibition. In addition, previous studies with βARKct and our present study with conditional GRK2 ablation provide strong evidence that GRK2 is the critical GRK regulating inotropic responsiveness and adverse βAR desensitization after MI. Interestingly, we found novel and significant evidence that β2-AR signaling is enhanced in GRK2 KO mice post-MI. Because β2-AR mediates protective effects likely through Gi and downstream activation of phosphatidylinositol 3-kinase and Akt,28,29 the enhanced β2-signal might, at least in part, contribute to the benefits seen with GRK2 silencing.
An Apparent Narrow Therapeutic Window for Altering βAR signaling in HF
GRK2 is a major regulator of myocardial βAR signaling, and dysfunctional βAR signaling is a consequence of HF and might, at least in part, contribute to the progression of cardiac dysfunction because failing human myocardium is characterized by diminished βAR responsiveness, loss of overall cardiac and single myocyte contractility, and disruption of intracellular Ca2+-cycling.27,30 Importantly, βAR derangements include the upregulation of GRK2.31,32 βAR stimulation in myocardium modulates intracellular Ca2+ fluxes and Ca2+ responsiveness of the sarcomere, thereby linking βAR signaling to cardiac myocyte contractility. Of note, chronic upstream activation of the βAR-signaling cascade by cardiac overexpression of the β1-AR2 or the adenylyl cyclase–stimulating G protein α-subunit (Gαs)4 led to the development of dilated cardiomyopathy in mice. Moreover, overexpression of protein kinase A, a downstream effector of βAR-Gs signaling also resulted in fatal dilated cardiomyopathy.33 Despite the evidence showing detrimental effects of chronic βAR and downstream activation in the heart, other studies manipulating βAR signaling have led to positive outcomes. This includes studies by us showing the prevention and rescue of HF using the βARKct,13,14 as well as overexpression of adenylyl cyclase VI, that has been shown to increase contractile function in the heart, as well as inhibiting the development of HF post-MI.34,35 Recently, β1-AR transactivation of the epidermal growth factor receptor mediated by β-arrestin was shown to confer cardioprotection in stressed hearts.36 Taken together, these genetic manipulations at different levels of βAR signaling demonstrate that the point of intervention is important to the function of the heart and suggest that it is more desirable to circumvent βAR desensitization than to simply facilitate βAR activation. Overall, the data discussed and our data suggest a narrow therapeutic window for βAR signaling in HF and indicate that normalization of βAR signaling, which can be best achieved by preventing their uncoupling and downregulation through GRK2 inhibition/ablation, might be the optimal approach, rather than forced overexpression of receptors or their downstream signaling effectors.
Protein kinases are rapidly emerging as a new pharmacological approach that may complement the existing drug classes targeting G protein–coupled receptors. Taken together, our present results show that targeted deletion and lowering of cardiac myocyte GRK2 activity leads to a novel protective and inotropic phenotype, which prevents postischemic HF and rescues a phenotype of established HF. Our results herein, combined with our previous βARKct data, add to the significance of the βAR system in the failing heart because it is clear that resensitization can positively affect cardiac function. We have not ruled out that other G protein–coupled receptors signals, no doubt altered by the loss of GRK2, are also contributing to the above phenotypes; however, the above-described changes in βAR inotropic reserve are significant. Overall, our data demonstrate that GRK2 activity is pathogenic in HF and targeted inhibition or lowering its expression led to beneficial effects for contractile function of the heart and this lone molecular change in the postischemic heart can prevent and rescue structural HF and offer novel benefits over existing HF therapies.
Sources of Funding
This work was supported, in part, by Deutsche Forschungsgemeinschaft grant Ra 1668/1-1 (to P.W.R.) and NIH grants R01 HL61690, R01 HL56205, and P01 HL075443 (Project 2) (to W.J.K.). W.J.K. is the W.W. Smith Professor of Medicine. The research performed by G.W.D. for this study was supported by NIH R01 HL87871.
↵*Both authors contributed equally to this work.
Original received November 20, 2007; revision received June 30, 2008; accepted July 3, 2008.
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