Physiological Induction of a β-Adrenergic Receptor Kinase Inhibitor Transgene Preserves β-Adrenergic Responsiveness in Pressure-Overload Cardiac Hypertrophy
Background—Transgenic mice with constitutive myocardium-targeted expression of a peptide inhibitor of the β-adrenergic receptor kinase (βARKct) have increased in vivo cardiac function and enhanced β-adrenergic receptor (βAR) responsiveness.
Methods and Results—In the present study, we created transgenic mice with myocardium-targeted βARKct transgene expression under control of the CARP (cardiac ankyrin repeat protein) promoter, which is active during cardiac development and inactive in the normal adult mouse heart. Consistent with this, adult CARP-βARKct transgenic mice have normal in vivo cardiac contractility and βAR responsiveness indistinguishable from their nontransgenic littermates (NLCs). However, because CARP is in a group of fetal genes activated in the adult ventricle during hypertrophy, we subjected animals to transverse aortic constriction (TAC) to induce pressure overload. Seven days after TAC, CARP-βARKct hearts had elevations in left ventricular mass similar to those in NLCs; however, TAC did induce demonstrable βARKct expression in the transgenic hearts. TAC in NLC mice resulted in an upregulation of myocardial βARK1 and a loss of βAR-mediated inotropic reserve. Importantly, although βARK1 was increased in the hypertrophic CARP-βARKct mice, the in vivo loss of βAR responsiveness was not seen after induced βARKct expression.
Conclusions—These results demonstrate that acute βARK1 inhibition can restore lost myocardial βAR responsiveness and inotropic reserve in vivo. Furthermore, these mice demonstrate the novel utility of the CARP promoter as an inducible element responsive to pathophysiological conditions in the adult heart.
Under normal physiological conditions, the β-adrenergic receptor (βAR) signaling system plays a pivotal role in the control of cardiac function, mediating the inotropic, chronotropic, and lusitropic responses to the sympathetic neurotransmitters epinephrine and norepinephrine.1 These receptors are in turn regulated by βAR kinase (βARK1), a member of the G protein–coupled receptor kinase (GRK) family that phosphorylates agonist-occupied receptors that trigger the binding of β-arrestins and the process of homologous desensitization.2 3 The action of the primarily cytosolic βARK1 (also known as GRK2) toward agonist-occupied receptors, such as βARs in the heart, is preceded by a membrane-targeting event that is accomplished by the direct interaction between dissociated βγ-subunits of activated G proteins (Gβγ) and residues within the carboxyl terminus of βARK1.4 5
βARK1 expression and activity in the heart appear to constitute a critical regulator of in vivo contractile function.6 7 Transgenic mice with myocardium-targeted overexpression of βARK1 have a loss of βAR-mediated inotropic reserve.6 Enhanced βARK1 expression and activity are present in the failing human heart8 and probably contribute to the well-known βAR pathology present in this disease.8 9 In addition to heart failure, enhanced βARK1 expression is responsible for the loss of myocardial βAR responsiveness in pressure-overload cardiac hypertrophy,10 and it has been shown to be increased in myocardial ischemia11 12 and hypertension.13 Importantly, reciprocal results (ie, enhanced in vivo cardiac function) have been found after the lowering of βARK1 activity in the heart.6 7 14 15 The activity of βARK1 can be inhibited in vitro and in vivo by a peptide composed of the Gβγ binding domain of βARK1 contained within the last 194 amino acid residues (βARKct).5 6 15 Interestingly, although complete knockout of the βARK1 gene leads to embryonic lethality,16 gene-targeted mice with a loss of 1 βARK1 allele have enhanced in vivo contractility.7 Thus, inhibiting the activity of βARK1 or lowering its expression appears to offer a novel means to enhance cardiac function.
The promoter used for these previous myocardium-targeted transgenic mice was the α-myosin heavy chain (αMyHC) gene promoter, which is not active in the mouse ventricle until around birth and then is constitutively active throughout adulthood.17 Due to the embryonic lethality found in βARK1 knockout mice, we sought to express the βARKct in the fetal heart to investigate potential cardiac requirements for βARK1 during development. To do this, transgenic mice were generated with βARKct expression targeted to the developing heart using the gene promoter for the cardiac ankyrin repeat protein (CARP). The CARP gene product is expressed early in cardiac development, detectable at embryonic day 7.5 to 8.0, and functions as a downstream negative regulator in the Nkx2-5 pathway.18 19 20 CARP levels remain high throughout cardiac development but decline 2 weeks postnatally, persisting at low or virtually undetectable levels in the adult heart.18 20 Transgenic mice harboring the CARP-lacZ transgene have shown that the promoter is developmentally regulated and induced as part of the embryonic gene program in response to hypertrophic stimuli.20 Therefore, in addition to driving βARKct expression in the developing heart, the present study used CARP-βARKct mice as a novel model demonstrating the utility of the CARP promoter to activate transgene expression acutely in the adult heart in response to a pathophysiological state.
The CARP-βARKct transgene was constructed by a 3-way ligation between the 2.2-Kb CARP promoter fragment (a generous gift from Drs Ken Chien and Ju Chen, University of California at San Diego), the 800-bp βARKct fragment, and the pGEM-SV40 plasmid containing the SV40 intron poly(A) signal.6 The resulting transgene construct was linearized and purified before pronuclear injections were performed by the Duke Comprehensive Cancer Center Transgenic Facility. Two lines of mice were established, CARP-βARKct-3 and CARP-βARKct-9. Litter sizes and postnatal development were indistinguishable from nontransgenic littermate controls (NLCs). Offspring were screened by standard polymerase chain reaction techniques with forward and reverse primers corresponding to specific regions of the CARP promoter and βARKct, respectively (Figure 1A⇓). Second-generation adult animals of both sexes (2 to 5 months of age) were used for all physiological studies. Institutional Review Board approval for all mouse experiments was obtained from Duke University.
In vivo pressure overload was created in the mouse by surgical banding of the transverse aorta as described previously.10 21 Transverse aortic constriction (TAC) causes pressure overload on the heart and reproducible cardiac hypertrophy.10 21 Sham-operated animals underwent the same surgical procedure without TAC. Operative mortality was <10% in both groups.
Hemodynamic Evaluation in Intact Anesthetized Mice
Initial in vivo hemodynamic evaluation to identify any functional phenotype was performed in anesthetized open-chest adult NLC and CARP-βARKct animals as described previously.6 Subsequently, hemodynamic parameters were evaluated 1 week after TAC in closed-chest anesthetized mice as described previously.14 Hemodynamic measurements were recorded at baseline and after a graded dose of isoproterenol (50, 500, and 1000 pg). After evaluation, hearts were rapidly excised and individual chambers separated, weighed for hypertrophy measurements, and frozen in liquid N2 for biochemical analysis.
Northern Blot Analysis
Hearts from the various experimental animals were excised as described above, and total RNA was isolated and Northern blot analysis performed by standard methods previously described.6
Immunodetection of myocardial βARKct expression was performed on cytosolic extracts prepared from homogenized NLC or CARP-βARKct hearts (with or without TAC) as described previously.6 10 Myocardial βARK1 levels were detected by immunoblotting for the ≈80-kDa βARK1 protein after immunoprecipitation from myocardial extracts with a βARK1 monoclonal antibody, as described previously.10 22
In Vitro GRK Activity Assays
Assessment of myocardial GRK activity was performed with cytosolic extracts and rhodopsin-enriched rod outer-segment membranes as described previously.10 22 The GRK activity found in cardiac cytosolic extracts is almost exclusively due to βARK1.10 [32P] incorporation into the rhodopsin substrate was quantified from dried gels with a Molecular Dynamics PhosphorImager.10 22
Data are expressed as mean±SEM values. Student’s t test was used to test for significance for parameters of hypertrophy, βARK1 expression, and GRK activity. To test differences in hemodynamic responses between groups with and without TAC, a repeated-measure ANOVA was used. For all analyses, P<0.05 was considered significant.
Generation and Characterization of CARP-βARKct Transgenic Mice
The 2.2-Kb proximal promoter region of the CARP gene has been previously reported to direct high-level expression of marker transgenes to the developing myocardium.18 20 In 2 individual lines of transgenic mice (designated lines 3 and 9), neonatal expression of the βARKct transcript was confined primarily to the heart, with minor mRNA expression seen in skeletal muscle and very little, if any, expression in other tissues (Figure 1B⇑). Cardiac expression of the βARKct transgene determined by Northern analysis and protein immunoblotting in neonates demonstrated equivalent expression in both lines (data not shown). Thus, all further experiments were performed on heterozygote CARP-βARKct-9 mice. Importantly, the expression of βARKct throughout the developing myocardium did not lead to any obvious pathology in neonates or adults.
A time course to monitor βARKct expression in the heart from birth to adulthood was performed. CARP-βARKct mice older than 1 month had significantly lower levels of myocardial βARKct expression by Northern analysis (Figure 2A⇓). Western blot analysis of the βARKct peptide produced similar results (Figure 2B⇓). In fact, by day 14, average βARKct mRNA and peptide expression in CARP-βARKct hearts was ≈50% of fetal (e18.0) cardiac levels, and by 30 days, very little, if any, transgene (mRNA or protein) expression was detected (Figure 2⇓). The loss of βARKct expression within the first 2 months of life is consistent with previously demonstrated properties of the CARP promoter.18 20
In vivo physiological assessment was made in adult mice. Consistent with low and undetectable levels of myocardial βARKct expression, in vivo cardiac function in adult CARP-βARKct mice was not different from corresponding NLCs. As shown in Figure 3⇓, basal and isoproterenol-stimulated left ventricular (LV) dP/dtmax responses measured in open-chest anesthetized mice were not altered in CARP-βARKct animals. Additional hemodynamic parameters were also found to be similar in NLC and CARP-βARKct mice (data not shown). Consistent with no change in βAR-mediated in vivo cardiac function, there was no difference in myocardial βAR density between adult NLC mice (21.0±3.4 fmol per mg membrane protein, n=4) and CARP-βARKct mice (25.7±2.1 fmol per mg, n=6) (P=NS). Thus, developmental expression of βARKct does not affect postnatal βAR density.
Pressure Overload in CARP-βARKct Mice
We subjected adult mice to LV pressure overload in vivo to determine whether the CARP-βARKct transgene could be induced. Previously, it has been shown that the endogenous CARP gene is included in a group of fetal genes, such as ANF and skeletal actin, that are silent in the adult ventricle but are activated during hypertrophy.20 Pressure-overload LV hypertrophy was induced by TAC, which we have previously used on mice.10 21 Seven days after TAC, mice develop a reproducible and stable LV hypertrophy that interestingly is accompanied by a loss of βAR responsiveness and inotropic reserve.10 As shown in Figure 4⇓, morphological and biochemical markers of pressure-overload hypertrophy were identical in both NLC and transgenic mice. Significant pressure-overload LV hypertrophy was achieved after 7 days of TAC as measured by an increase in LV/body weight (BW) ratio in both CARP-βARKct and NLC mice (Figure 4A⇓). The significant increase in LV/BW ratio was on the order of 43% to 54%, and the TAC-induced increases in LV mass were not statistically different between CARP-βARKct and NLC animals. The increase in LV/BW ratio was mirrored by an increase in βARK1 expression after TAC, as measured by protein immunoblotting (Figure 4B⇓ and 4C⇓). Consistent with increased βARK1 protein levels, basal myocardial GRK activity was also significantly enhanced after TAC, and this increased kinase activity in the absence of Gβγ (≈3-fold) was similar in CARP-βARKct and NLC mice (data not shown).
Myocardial Physiological Properties After Hypertrophic Induction of βARKct
To determine the in vivo physiological response to TAC and hypertrophy, cardiac catheterization was used to measure βAR responsiveness in intact anesthetized TAC and sham-treated NLC and CARP-βARKct mice (Figure 5⇓). In these studies, anesthetized mice were analyzed in a closed-chest model, which differs from the open-chest measurements obtained for Figure 3⇑. In response to isoproterenol, LV dP/dtmax in NLC mice was significantly blunted after TAC, which is consistent with enhanced βARK1 activity. Interestingly, although CARP-βARKct animals also have hypertrophy and enhanced βARK1 expression after TAC, their βAR-mediated cardiac contractility is preserved and, in fact, significantly enhanced compared with NLC-TAC mice (Figure 5⇓). Moreover, basal LV dP/dtmax in CARP-βARKct-TAC mice is significantly higher than in NLC-sham mice (Figure 5⇓). The Table⇓ includes all hemodynamic parameters measured in these mice, including LV dP/dtmin as an index of cardiac relaxation, heart rate, LV systolic pressure, and LV end-diastolic pressure. Interestingly, NLC-TAC mice have a limited relaxation (LV dP/dtmin) response after isoproterenol administration; however, diastolic βAR responsiveness is restored in CARP-βARKct mice after pressure overload (Table⇓). The differences between groups for LV dP/dtmin were found to be significant (Table⇓).
Northern blot analysis of RNA isolated from CARP-βARKct hearts that had undergone TAC showed the appearance of βARKct mRNA, which was absent in sham-operated age-matched CARP-βARKct animals (Figure 6A⇓). Importantly, this induction of transgene mRNA expression was confirmed by protein immunoblotting (Figure 6B⇓), which demonstrated the appearance of detectable levels of the βARKct peptide in myocardial extracts after TAC in CARP-βARKct animals. As shown in Figure 6B⇓, there was some variability in the induction of βARKct expression after TAC. In addition to TAC, we found induction of βARKct expression in the hearts of adult CARP-βARKct mice after chronic infusion (7 days) of isoproterenol (data not shown) at doses sufficient to cause myocardial hypertrophy.22 23
The present study reports 2 novel findings, including the hypertrophic induction of cardiac βARKct expression in the adult heart when driven by the CARP promoter. Second, this is the first report of the activity of βARK1 being inhibited in the developing mouse ventricle. The results of this study demonstrate that induction of βARKct in the hypertrophied adult heart is able to block myocardial βAR desensitization associated with pressure-overload hypertrophy and preserve in vivo inotropic reserve. These results are similar to what has been described previously in αMyHC-βARKct mice in which βARKct was constitutively expressed in the adult heart.10 However, the present study demonstrates that induced βARKct expression in the heart can acutely preserve normal βAR-mediated cardiac signaling and function even in the face of enhanced βARK1 expression and GRK activity.
Increased βARK1 expression and activity characterize many forms of cardiac hypertrophy and disease. However, βARK1 upregulation is not a generalized biochemical marker of hypertrophy because it is not observed in transgenic models of hypertrophy induced by cardiac overexpression of oncogenic ras,10 or Gαq.24 Enhanced βARK1 expression in the diseased heart may be triggered by heightened sympathetic nervous system activity, which is present in most cardiovascular disorders as an early compensatory mechanism. This hypothesis has been bolstered by recent studies in the mouse22 23 in which chronic infusions of the βAR agonist isoproterenol resulted in increased mRNA, protein, and activity of βARK1 in the heart. However, this was not seen after phenylephrine-induced hypertrophy, which indicates that catecholamine induction of βARK1 expression in the heart occurs exclusively via stimulation of βARs and not α-adrenergic receptors.23
Pressure-overload hypertrophy in vivo has been demonstrated recently to be triggered by signals that activate receptors coupled to the G protein Gq, because targeted inhibition of the myocardial receptor–Gq interface in transgenic mice attenuated myocardial hypertrophy after TAC.21 However, it is believed that TAC will increase sympathetic nervous system activity; thus, βARK1 may be elevated in the heart by catecholamines acting through βARs during the induction of pressure-overload cardiac hypertrophy via Gq-coupled receptors.
Interestingly, the level of βARKct expression after TAC did not appear to consistently reach the levels found in the previously described αMyHC-βARKct transgenic mouse (Figure 6⇑). However, this apparently lower βARKct expression induced in CARP-βARKct mice was sufficient to preserve myocardial βAR responsiveness after hypertrophy. These results represent an important advance in the therapeutic potential of βARK1 inhibition, because previous data demonstrating the reversal of βAR desensitization in hypertrophy10 and the prevention of cardiomyopathy in a murine model of heart failure due to MLP(−/−) gene ablation14 were accomplished by αMyHC-βARKct transgenesis in which βARKct was expressed in the adult ventricle from birth. Accordingly, it will be interesting to determine in future studies whether CARP-βARKct mice with inducible βARK1 inhibition can also “rescue” mouse models of heart failure, including longer-term follow-up after TAC, when decompensation and cardiac dysfunction can occur.
This study also represents the first report of developmental cardiac expression of βARKct in transgenic mice. Importantly, there were no overt alterations in the size or function of the heart due to fetal ventricular βARKct expression driven by the CARP promoter. These results actually are consistent with the hearts of heterozygous βARK1 knockout mice that develop normally.7 16 An aspect of fetal cardiac βARKct expression that might present a problem in development involves other Gβγ-dependent processes. We have previously shown that Gβγ can activate the p21ras–mitogen-activated protein kinase pathway25 and have recently used βARKct to inhibit pathological in vivo vascular smooth muscle cell proliferation.26 27 28 Thus, βARKct has the potential to inhibit any role Gβγ may have in the developing heart. Our results with myocardial βARKct expression throughout gestation suggest that Gβγ does not play a major role in developmental cardiac mitogenesis, which in itself is a significant finding.
A potential novel application of the results of this study concerns gene therapy. In development of future cardiac gene transfer vectors (adenoviral or adeno-associated virus), the CARP promoter may offer advantages over other cardiac promoters. The CARP promoter not only targets transgene expression primarily to the heart but also will only be active during pathophysiological states. For example, a therapeutic transgene could be delivered to the failing heart to produce a beneficial effect, and if the disease process stops, the transgene will also be eliminated via the functional “turnoff” of the CARP promoter. However, transgene expression could be reestablished if the need arises, such as a relapse of the cardiac pathology. This could be a powerful application of βARKct, because it appears that the acute inhibition of βARK1 is a novel therapeutic target in conditions of compromised heart function. Importantly, this can be accomplished either by a gene therapy approach or the development of small molecules that can inhibit βARK1 activity.
This work was supported in part by National Institutes of Health grants HL-61690 (Dr Koch) and HL-56687 (Dr Rockman).
- Received May 25, 2000.
- Revision received June 21, 2000.
- Accepted June 22, 2000.
- Copyright © 2000 by American Heart Association
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