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Cardiovascular Research 2001 50(3):443-453; doi:10.1016/S0008-6363(01)00244-9
© 2001 by European Society of Cardiology
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Copyright © 2001, European Society of Cardiology

Sympathoadrenergic mechanisms in functional regulation and development of cardiac hypertrophy and failure: findings from genetically engineered mice

Xiao-Jun Du*

Experimental Cardiology Laboratory, Baker Medical Research Institute, P.O. Box 6492, St. Kilda Road Central, Melbourne, Victoria 8008, Australia

* Tel.: +61-3-9522-4333; fax: +61-3-9521-1362 xiaojun.du{at}baker.edu.au

Received 2 August 2000; accepted 12 February 2001

KEYWORDS Autonomic nervous system; Cardiomyopathy; Heart failure; Hypertrophy; Receptors; Signal transduction


   1 Introduction
Top
 1 Introduction
2 Phenotypes of mice...
 3 Phenotype of strains...
 4 Comparison of cardiac...
 5 Presynaptic factors...
 6 Gene complementation
 7 Conclusions
 References
 
Sympathoadrenergic signals to the heart are detected and processed by a group of membrane proteins converting external signals to alterations in intracellular properties. In recent years, our understanding of β- and {alpha}-adrenergic signaling has been significantly improved. It is beyond the scope of this review to discuss the adrenergic signaling pathways and several excellent reviews are available [1–5].

Sympathetic activation serves to maintain cardiac output by enhancing inotropy, relaxation and chronotropy. Under conditions of heart failure (HF), the sympathetic nervous system is activated chronically and enhanced cardiac sympathetic drive frequently precedes the onset of overt clinic symptoms and is one of the fundamental abnormalities in HF [6]. In the hypertrophied and failing heart, there occurs profound desensitization of β-adrenergic receptor (AR) signaling due to decreased β1AR density, uncoupling of βAR to stimulatory G-protein (Gs), diminished adenylyl cyclase (AC) activity, increased βAR kinase (βARK) activity, and elevated content and function of inhibitory G-protein (Gi) [1,4,7]. The pathophysiological implications of these alterations are only partially understood.

Numerous mouse strains have recently been created that target key components in the signaling pathways as well as these components involved in sympathetic activity (Table 1) [8–47]. These genes are either overexpressed in the heart, mostly under the {alpha}-myosin heavy chain promoter, or are disrupted or modified. These strains provide us with systematic tools and novel opportunities to address a number of fundamental questions crucial to our understanding of the contribution by the sympathoadrenergic system to cardiac physiology and pathophysiology. These questions include the difference in signaling mediated by β1AR and β2AR, roles of βARK1 in βAR signaling and functional regulation, activities of {alpha}1AR subtypes in cardiac function and hypertrophic growth, contributions of heterotrimeric G-proteins (like Gq and Gi) to the development of hypertrophy and HF, and the potential of upregulating β-adrenergic signaling in HF by gene therapy. These questions, difficult or impossible to answer using conventional methodology, are being addressed using gene-targeted mice in vivo. Accumulating data from research using murine strains have significantly advanced our knowledge in this area. However, because adaptational changes secondary to a gene overexpression or disruption have been explored little, caution is required when attributing all phenotypes to the direct consequences of the alterations of a targeted protein.


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  		Table 1 Genetically engineered mouse models targeting the sympathoadrenergic signaling system

 

   2 Phenotypes of mice with β- or {alpha}-adrenergic receptor disruption or overexpression
Top
 1 Introduction
 2 Phenotypes of mice...
3 Phenotype of strains...
 4 Comparison of cardiac...
 5 Presynaptic factors...
 6 Gene complementation
 7 Conclusions
 References
 
Almost all subtypes of ARs have been genetically manipulated producing various strains of mice. Studies on their cardiac phenotypes have yielded important insights into the physiological and pathophysiological roles of specific receptor subtypes.

2.1 Disruption of βAR
The role of βARs in controlling cardiac physiology has been assessed by individual or combined disruption of genes coding for β1AR, β2AR or β3AR [9,13,15–17]. Mice lacking β1AR can survive to adulthood without any gross abnormalities. Baseline heart rate, blood pressure, heart weight and myocardial content of norepinephrine are similar to those seen in wild-type littermates. Increases in AC activity and contractile force in responses to isoproterenol were absent in β1AR deficient mice whilst the response to forskolin remained intact [9]. These results suggest that β1AR is the main subtype of βARs mediating responses to catecholamine stimulation in vivo while the contribution of β2AR is minor, a finding supported by the unaltered cardiac function at baseline and during β-agonist stimulation in β2AR knockout mice [13]. Surprisingly, despite the total absence of βAR, β1/β2 null mice generated by cross-breeding had similar resting heart rate, blood pressure and maximal exercise capacity as wild-type mice. However, these mice did have reduced ventricular contractility and relaxation estimated from the lower rates of left ventricular (LV) pressure increase and fall (dP/dt), and a lower oxygen consumption during exercise [17]. Mice lacking β3AR had an increased fat storage [15,16] but their cardiac phenotype has not been reported.

In summary, the phenotypes observed from βAR knockout models support the view that under basal conditions, a fully functional β-adrenergic system is not essential to achieve normal function for a healthy heart. However, this system is essential for supporting circulatory function under stress such as exercise [17]. More thorough studies involving application of a variety of stressors or disease, and longitudinal follow-up of cardiac function in these lines should provide useful information regarding the β-adrenergic mechanisms involved in regulation of cardiac function and onset of HF.

2.2 Overexpression of βAR
2.2.1 β1AR
Mouse strains overexpressing wild-type β1AR were reported with a 5–15-fold increase in receptor density [8]. These mice at very young age displayed higher levels of baseline heart rate and LV contractility, as well as enhanced inotropic and chronotropic responses to a β-agonist isoproterenol, confirming an enhanced β1AR signaling. Cardiomyopathy evidenced by myocyte hypertrophy and interstitial fibrosis, was evident at 8–16 weeks of age, and LV dysfunction and immature death occur around 20 weeks [12]. These findings were confirmed by another group in a strain of mice with β1AR increased by 20–40-fold [48]. Thus, modest β1AR overexpression leads to an early occurrence of cardiac pathology and dysfunction.

2.2.2 β2AR
Mice that overexpress wild-type β2AR by 100–350-fold in the heart exhibit markedly enhanced cyclic AMP (cAMP) content and myocardial contractility [10,11]. This agonist-independent β2AR activation is explained by existence of the active conformer of β2-AR. Whereas the inactive conformer is by far the predominant one, with >100-fold increase in β2AR density, the number of active receptors would be substantially higher and sufficient to support a full physiological stimulation [10]. In the mice with markedly increased β2AR density, the functional response to β-agonist is largely lost [3,10,11,49]. Recent studies have revealed that the unresponsiveness to β2-agonists is due to tight coupling of β2AR to Gi by which Gs-mediated responses are minimized and localized [3]. The concomitant lack of response to β1AR stimulation in these mice is likely due to the heterogeneous desensitization of β1AR, independent of βARK1 or Gi [50], or competition with β1AR for Gs by the overexpressed β2AR [51].

The markedly enhanced myocardial contractility in β2AR-overexpressing mice is consistent with the view that β2AR overexpression in the myocardium might be an approach for HF gene therapy. Indeed, in vivo and in vitro transfection with β2AR gene preserved β-adrenergic signaling and improved cardiac function [52]. However, a key question is whether a long-term β2AR activation may lead to adverse consequences. We have studied a cohort of mice overexpressing β2AR by 300-fold [10] by performing echocardiography from 4 to 15 months of age. Within 6 months wild-type and β2AR-overexpressing mice had similar LV size and fractional shortening. After 9 months, however, β2AR-overexpressing mice exhibited progressive ventricular dilatation with reduced fractional shortening and systolic wall thickening [53]. By 15 months the cumulative mortality was 81% in the transgenic mice, mainly due to HF. Hearts from these mice displayed reduced number of cardiomyocytes, widespread fibrosis and severe hypertrophy [53]. Liggett et al. [11] demonstrated that mice overexpressing β2AR at higher levels (100- and 350-fold) developed fibrotic cardiomyopathy and early death, but mice with 60-fold β2AR exhibited higher basal ventricular function but had no obvious abnormalities at 12 months of age. Thus, the consequence of enhanced β-adrenergic activity in the heart is dependent on the level of transgene overexpression. Although long-term β2AR overexpression at very high levels is deleterious with cardiomyopathic phenotype being similar to that reported in young mice overexpressing β1AR [8,48], modest β2AR overexpression enhanced basal and stimulated myocardial contractility with no pathological consequences.

Polymorphisms of β2AR, such as Ile164 and Gly16, have been described in humans. Turki et al. [12] reported a transgenic line that expresses β2AR with a point mutation (Ile164Thr) at the 4th transmembrane domain where the β2AR couples to Gs. Overexpression of the Ile164 β2AR by 45-fold had no deleterious effects. However, compared to another transgenic line overexpressing equivalent amount of wild-type β2AR, these mice had lower levels of heart rate, LV dP/dt and AC activity at baseline and during β-agonist stimulation, indicating the defect of Ile164 β2AR in coupling to Gs [12]. The phenotypes of Ile164 β2AR mice provide strong evidence that such β2AR polymorphism is functionally relevant and bears pathophysiological significance. In clinic setting, although β2AR polymorphisms per se do not lead to HF, HF patients with such polymorphisms show a reduced exercise performance and are at higher risk for rapid decompensation [54,55].

2.2.3 β3AR
In mice overexpressing β3AR at 1.6 pmol/mg protein, β3AR stimulation markedly enhanced the ventricular contractility and activated AC [14], indicating coupling of β3AR to Gs. In contrast, the wild-type mice showed no response to such stimulation.

2.3 {alpha}AR overexpression or disruption
Two subtypes of {alpha}1AR, {alpha}1A and {alpha}1B, exist in the heart as functional proteins. Stimulation of {alpha}1AR activates Gq pathway leading to phosphatidylinositide hydrolysis and release of intracellular Ca2+. While acute stimulation of {alpha}1AR has limited influence on cardiac function under physiological conditions, activation of {alpha}1AAR/Gq pathway has been implied as a key mechanism for cardiac hypertrophic growth [4,5].

2.3.1 {alpha}1AAR
Lin et al. [18] developed a strain of mice with cardiac-specific overexpression of {alpha}1AAR. These mice with over 100-fold increases in {alpha}1AAR density display markedly increased LV dP/dtmax but only a minor increase in dP/dtmin with the dP/dtmax:dP/dtmin ratio increased to 1.9 from 0.9 seen in wild-type controls, suggesting a selective enhancement of inotropy but not relaxation. LV fractional shortening was also significantly increased in {alpha}1AAR overexpressing mice. With dP/dtmax already enhanced in these mice, isoproterenol was unable to increase dP/dtmax further. The mechanism for this potent inotropic effect by {alpha}1AAR overexpression remains unclear. In contrast to the view of a key role of {alpha}1AAR in the development of hypertrophy, these mice developed no sign of cardiac hypertrophy [18]. Although atrial natriuretic factor (ANF) mRNA increased by 7-fold, expression of other hypertrophy-associated genes, such as myosin light chain 2v, β-myosin heavy chain, and skeletal {alpha}-actin, remain unchanged [18].

2.3.2 {alpha}1BAR
Two strains have been developed overexpressing either wild-type or active mutant {alpha}1BAR [19,20]. Mice overexpressing active mutant {alpha}1BARs developed mild hypertrophy with a 20% increase in heart weight and several fold upregulation of hypertrophy-associated genes [20,56]. Cardiac hypertrophy in this strain is mediated by Gq as concomitant expression of a Gq inhibitory peptide in these mice inhibits the development of cardiac hypertrophy [57]. We observed that the active mutant {alpha}1BAR mice with pressure overload developed more severe hypertrophy despite the fact that {alpha}1AAR expression and activity were markedly suppressed [56]. In mice with cardiac overexpression of the wild-type {alpha}1BAR by 26–43-fold [19], there was an 8-fold increase in ANF mRNA levels but no sign of cardiac hypertrophy. Interestingly, although βAR density remained unchanged, these mice had attenuated inotropic and chronotropic responses to isoproterenol and suppressed AC activity [19,58]. These abnormalities can be reversed by inactivation of Gi with pertussis toxin suggesting that stimulation of {alpha}1BAR suppresses β-adrenergic signaling via activation of Gi [19,59]. Another possibility is activation of the Gq/protein kinase-C (PKC) pathway with subsequent activation of βARK1 and downregulation and uncoupling of β1AR [5,59–61]. Indeed, three PKC isoforms ({delta}, {varepsilon} and β) were elevated and βARK1 was increased by 3-fold in hearts overexpressing {alpha}1BAR [19,61]. Compared with nontransgenic littermates, {alpha}1BAR transgenic mice treated with phenylephrine for 14 days displayed more severe hypertrophy, a higher βARK1 activity and poor survival (20 versus 90%) [61]. Disruption of {alpha}1BAR suppressed hypertensive or vasoconstrictive responses to {alpha}-agonist stimulation and reduced total {alpha}1AR density in the heart by 74% [21]. The functional consequence to the heart has not been investigated.

2.3.3 Summary
There are several novel discoveries from the studies outlined above. First, whereas {alpha}1AR stimulation in cultured rat cardiomyocytes promotes hypertrophy, there is no evidence that activation of {alpha}1AAR or {alpha}1BAR by cardiac overexpression mediates hypertrophic growth in vivo. This questions the role of {alpha}1AR in the development of cardiac hypertrophy in vivo. Obviously, it remains to be investigated as to whether the pro-hypertrophic effect of {alpha}1AR is more evident under pathological conditions, such as pressure overload or long-term catecholamine administration. Second, cardiac-directed overexpression of {alpha}1AR subtypes leads to distinct functional phenotypes with enhanced inotropy in {alpha}1AAR transgenic mice but suppressed contractility in mice that overexpress {alpha}1BAR. These opposite functional outcomes in mice overexpressing {alpha}1AAR and {alpha}1AAR strongly indicate certain important differences in the signal pathways regulating myocardial function although both receptor subtypes couple to Gq. Third, activation of {alpha}1BAR has negative inotropic consequence and potently suppresses β1AR signaling. These effects most likely pertain to the failing heart as the density and signaling of {alpha}1ARs are well maintained under this situation [4]. Further research on these mouse models also needs to explore the role of {alpha}1AR as mediator for malignant arrhythmias.


   3 Phenotype of strains overexpressing downstream components of adrenergic signaling pathways
Top
 1 Introduction
 2 Phenotypes of mice...
 3 Phenotype of strains...
4 Comparison of cardiac...
 5 Presynaptic factors...
 6 Gene complementation
 7 Conclusions
 References
 
Functions of several G-proteins and effector enzymes of the adrenergic signal pathways have been intensively investigated using gene-targeted mouse models.

3.1 AR-coupled G-proteins
3.1.1 Gs{alpha}
Gaudin et al. [22] developed a strain showing a 5-fold overexpression of the Gs{alpha} subunit in the heart. These mice within 12 months of age displayed a higher baseline heart rate, enhanced cardiac function and augmented inotropic and chronotropic responses to β-agonist stimulation [62]. These observations are in keeping with a facilitated signaling via βAR coupling to Gs{alpha}. At 15 months of age, mice overexpressing Gs{alpha} developed cardiac abnormalities, including myocyte apoptosis, necrosis, hypertrophy, interstitial fibrosis, cardiac dilatation and dysfunction. These mice also had blunted heart rate variability and arterial baroreflex sensitivity and a mortality of 45% by 1.5 years of age [62–65]. Long-term β-blockade with propranolol reversed all these abnormalities [65].

3.1.2 Gi
Among the three subtypes of Gi{alpha}, Gi{alpha}2 is the most abundant in the heart. Mouse lines with either knockout of Gi{alpha}2 and Gi{alpha}3 or expression of inducible antisense Gi{alpha}2 have been generated [24–26]. In mice with cardiac-specific disruption of Gi{alpha}2 and Gi{alpha}3, baseline cardiac function and responses to isoproterenol were similar to wild-type mice [25]. It is well known that stimulation of Gi-coupled muscarinic receptor (M2) attenuates the βAR signaling [1]. In embryonic hearts lacking Gi{alpha}2, inhibition of isoproterenol-stimulated AC activity by a M2-agonist carbachol was markedly attenuated [24]. Redfen et al. [23] recently reported a strain with conditional expression of modified {kappa}-opioid receptors, Ro1. Modification of Ro1 prevents binding to and signaling of Ro1 by endogenous ligands. However, a synthetic agent spiradoline can effectively bind to Ro1 leading to potent activation of Gi signaling. When Ro1 is overexpressed and spiradoline administered, Ro1-overexpressing mice display dilated cardiomyopathy, intraventricular conduction delay and poor survival [23]. DNA-array analysis revealed changes largely restricted to the downstream of the Gi signal pathway together with a upregulated collagen expression [23]. These findings indicate the deleterious action of an enhanced Gi activity seen in the failing heart. Gi activation antagonizes βAR-mediated responses and this mechanism may contribute to cardiac dysfunction in Ro1 mice. However, the effects of other Gi-coupled receptors, like opiates, endothelin (ETA), acetylcholine (M2), adenosine (P1), angiotensin II (AT1) and histamine (H1), in the development of the phenotypes seen in Ro1 mice are unexplored.

3.1.3 Gq{alpha}
The mechanisms mediating hypertrophic growth and progression into failure are poorly understood. Gq is likely a key factor in these processes because activating diverse Gq-coupled receptors, including {alpha}1AR, AT1 and ETA, initiates myocardial hypertrophy [5]. Whereas no change in phenotype was observed in mice with 2-fold expression of wild-type Gq{alpha} subunit, the mice with 4–8-fold increase in Gq{alpha} in the heart displayed hypertrophy, enhanced expression of hypertrophy-associated genes, and exhibited a 2.6-fold increase in total PKC activity. These mice also developed LV dilatation and suppressed ventricular contractility. Whereas βAR density was unchanged, isoproterenol-stimulated AC activity was reduced by 70% in conjunction with attenuated functional response [27,66]. Histologically, interstitial fibrosis and apoptotic myocyte death were evident [66]. In another strain with modest expression (30–56% increase at protein level) of an active mutant Gq{alpha} subunit [28], the mice at a few weeks of age developed massive cardiac chamber dilatation, hypertrophy and fibrosis [28]. When exposed to aortic constriction, G{alpha}q-overexpressing mice displayed LV dilatation and HF whilst nontransgenic mice exhibited concentric LV hypertrophy with maintained ejection performance [67]. Cardiac-specific expression of a carboxyl-terminal peptide of Gq{alpha} subunit, Gqi, abolishes Gq-mediated signaling by inducing a dominant-negative effect and the Gqi mice with aortic constriction developed much less severe cardiac hypertrophy than controls [29]. Thus, these findings confirm a key role of Gq in mediating hypertrophy, apoptosis and cardiomyopathy and imply the inhibition of G{alpha}q signaling as a new therapeutic strategy. In accordance, overexpression of PKC-β results in hypertrophy, enhanced expression of hypertrophy-related genes and cardiomyopathy [34,35].

3.1.4 Gh
Gh, also known as transglutaminase II (TGII), binds and hydrolyses GTP and is believed to mediate {alpha}1AR-stimulated activation of PLC{delta}1 and phosphoinositide turnover [68]. However, mice with 6–37-fold increase in cardiac TGII activity did not show an increased PKC activity at baseline or with {alpha}1-agonist stimulation [69]. This is in sharp contrast to the Gq{alpha}-overexpressing mice showing markedly increased PKC activity and phosphoinositide turnover under these conditions [27,66]. Therefore, the data do not support a coupling of Gh to {alpha}1AR and activation of PKC by this pathway. Interestingly Gh-overexpressing mice developed myocardial hypertrophy, widespread interstitial fibrosis and exhibited suppressed LV function [69]. It has been suggested that TGII, via catalyzing the formation of protein cross-links, involves in regulation of cell growth, matrix formation and apoptosis. The phenotypes observed suggest a pathological role of an enhanced TGII activity in the heart.

3.2 Adenylyl cyclase
At least ten isoforms of AC have been identified. The isoforms ACV, ACVI and ACVIII are the most abundant in the heart. Mice with cardiac-directed overexpression of ACV and ACVI by 20-fold possess very similar phenotypes [31–33]. Transgenic and wild-type mice have similar heart rate, echocardiographic parameters, βAR density and content of Gs or Gi. Upon β-agonist stimulation, the transgenic mice showed more pronounced increases in LV contractility and cAMP content compared to control littermates [31,32]. Mice that overexpress Ca2+-activated ACVIII had several fold increases in AC and PKA activities, heightened intrinsic ventricular contractility and were unresponsive to β-agonist stimulation [33]. Interestingly, unlike β1AR, β2AR and Gs{alpha} transgenic lines, the ACVI-overexpressing mice do not develop morphological or functional abnormalities in the heart up to 20 months of age [31]. The lack of cardiac pathology in ACVI mice despite the markedly enhanced β-adrenergic signaling is unclear in the mechanism but seems to indicate some adverse consequences by Gs-independent signals following βAR overexpression.

3.3 βAR kinase-1 and β-arrestins
3.3.1 βARK1
βAR rapidly loses its responsiveness in the continued presence of agonists; a phenomenon called desensitization. The recognition of blunted β-adrenergic responsiveness to catecholamines in the failing heart has stimulated research to determine the underlying molecular events [1,4,7]. G-protein coupled receptor kinases (GRK) phosphorylate activated βARs leading to homologous desensitization. βARK1 (or GRK2) is one of six members of GRKs and the most important member in the heart [2]. The role of βARK1 has been thoroughly investigated in recent years by Lefkowitz, Koch and colleagues using gene-targeted approach [36,37,70,71]. Three models have been developed: cardiac-restricted overexpression of βARK1, cardiac expression of a βARK1 inhibitory peptide (βARKct) and βARK1 knockout [36,37]. Disruption of the βARK1 gene is embryonically lethal and βARK1-deficient embryos displayed marked ‘hypoplasia’ with thin cardiac chamber walls, enlarged chamber sizes and a profound reduction in LV ejection fraction [37], indicating the necessity of βARK1 for cardiac development. Heterozygous mice develop normally and, at maturity, display an approximately 60% reduction in βARK1 activity with enhanced LV contractility at baseline and during β-agonist stimulation [70]. In accordance, βARK1-overexpressing mice displayed attenuated inotropic response to isoproterenol although baseline LV contractility was similar to that of wild-type littermates [36]. βARKct is a peptide corresponding to the carboxyl-terminal of βARK1 and blocks interactions of Gβ{gamma} subunit and βARK1 thereby preventing the activation of βARK1 [2,37]. βARKct overexpression partially inhibited βARK1 activity and increased LV contractility at baseline and during βAR stimulation [37]. Whereas βARK1 overexpession lead to depressed AC activity and LV contractility, these abnormalities were normalized by concomitant expression of βARKct in the βARK1-overexpressing mice [71]. Improvement of LV contractility under basal and βAR stimulated conditions was also observed in mice with βARKct expressed acutely following pressure overload [72]. These observations confirm the cardiac depressing effect of enhanced βARK1 activity through desensitization and downregulation of β-adrenergic signaling and raise the possibility of βARK1 inhibition being a treatment strategy for improving myocardial contractility when cardiac function is compromised. This hypothesis remains to be tested using specific βARK1 inhibitors.

3.3.2 β-Arrestins
β-Arrestins consist of a family of four proteins, involved in the desensitization of G-protein-coupled receptors by binding with phosphorylated receptors [2]. β-Arrestin-1 and β-arrestin-2 are expressed in the heart and β-arrestin-1 is more abundant. Mice lacking β-arrestin-1 appear normal and, upon stimulation with isoproterenol, display enhanced functional responses. This is in keeping with the removal of the βAR inhibition mediated by this protein [38]. β-arrestin-2 disruption resulted in marked augmentation and prolongation of the analgetic effect mediated by the Gi/o-coupled µ-opioid receptor, indicating the lack of desensitization of this receptor in the absence of β-arrestin-2 [39]. The cardiac phenotype in these mice merits further investigation.


   4 Comparison of cardiac phenotypes of β-adrenergic signaling targeted strains
Top
 1 Introduction
 2 Phenotypes of mice...
 3 Phenotype of strains...
 4 Comparison of cardiac...
5 Presynaptic factors...
 6 Gene complementation
 7 Conclusions
 References
 
The transgenic lines overexpressing βARs and down-stream components share some similar phenotypes but also show several important differences.

Three transgenic lines, β1AR, β2AR (>100-fold) and Gs{alpha}, are similar in their development of fibrotic cardiomyopathy, LV dysfunction and failure [8,10,11,48,62–65], providing strong evidence for the pathological role of chronic and uncontrolled β-adrenergic activation in the development of HF. However, some important differences exist among these lines. Mice with 15–40-fold expression of β1AR developed severe cardiomyopathy and dysfunction within 2–4 months of age [8,48]. In contrast, β2AR expression at equivalent levels (30–60-fold) does not lead to obvious cardiac abnormalities [12] and overexpression at very high levels results in cardiomyopathy that occurs much later than that in β1AR overexpressing mice [11,53]. Such distinct phenotypes of β1AR versus β2AR transgenic strains imply fundamental differences in their signaling mechanisms [3]. Recent studies have revealed several differences in cardiac signaling. Both β1AR and β2AR activate AC via stimulation of Gs. Compared with β1AR, however, biochemical and functional responses following stimulation of β2AR are limited both spatially and temporarily [3]. β2AR, but not β1AR, couples to Gi and activation of Gi antagonizes Gs-mediated signaling [3]. Gi-coupling by β2AR also appears to be responsible for the inhibition of myocyte apoptosis. This again differs from a pro-apoptotic effect of β1AR stimulation or Gs{alpha} overexpression [63,73]. Furthermore, the bradycardiac effect mediated by Gi-coupled M2 is enhanced in β2AR-overexpressing mice [74] but markedly attenuated in transgenic lines expressing Gs{alpha} or β1AR in the atria [64,75].

Interestingly, mice overexpressing AC or βARKct appear to be free of cardiac abnormalities although enhanced β-adrenergic signaling is evident [31,36]. In addition, higher baseline heart rates exist in β1AR, β2AR and Gs{alpha} transgenic lines [8,10,11,48,49,53,62,64] but not in mice overexpressing AC or βARKct [31–33,36,70]. As chronic rapid pacing leads to HF in larger species, a higher basal heart rate might contribute to cardiac abnormalities in mice with βAR or Gs{alpha} overexpressed. Despite a similar elevation in basal ventricular contractility in mice overexpressing β2AR or βARKct, only β2AR mice display an exaggerated myocardial injury when exposed to ischemia/reperfusion [76]. Thus, a lifetime enhancement of β-adrenergic activity is not always associated with increased risk of cardiac abnormalities and that enhanced expression of effectors such as AC or βARKct, may bypass the deleterious consequences due to βAR or Gs overexpression.


   5 Presynaptic factors controlling sympathetic nervous activity
Top
 1 Introduction
 2 Phenotypes of mice...
 3 Phenotype of strains...
 4 Comparison of cardiac...
 5 Presynaptic factors...
6 Gene complementation
 7 Conclusions
 References
 
Several strains targeting the presynaptic factors of the sympathetic nervous system, have been generated with either enhanced or diminished sympathetic nervous activity.

5.1 Nerve growth factor (NGF) overexpression
NGF is an effector cell-derived neurotrophin and essential for the survival and growth of sympathetic neurons during embryonic development and maintenance of the noradrenergic phenotype. Indicative of pronounced sympathetic innervation, mice with cardiac-specific overexpression of NGF have higher sympathetic nerve density, a 22-fold increase in norepinephrine content and enhanced capacity of neuronal norepinephrine uptake [40,77,78]. Interestingly, β1AR downregulation, usually seen with heightened sympathetic stimulation, was not observed and β2AR density was actually increased by 2.3-fold. However, responses to isoproterenol as measured by increase in membrane Ca2+ current and AC activity, were diminished whereas activation of AC stimulated by Gpp(NH)p or forskolin remained intact [77]. This latter finding implies uncoupling of βARs in NGF-overexpressing hearts. Other phenotypic features include a 50% increase in heart/body weight ratio, myocyte hyperplasia and interstitial fibrosis [40,78].

5.2 Disruption of enzymes for catecholamine synthesis and metabolism
Biosynthesis of catecholamines by nerve varicosities and chromaffin cells requires hydroxylation of tyrosine by tyrosine hydroxylase (TH) and β-hydroxylation of dopamine by dopamine-β-hydroxylase (DβH). Disruption of the TH or DβH genes is embryonically lethal with only 5% newborns reaching adulthood [41,42]. Thus, catecholamines are essential for embryo development and loss of functional support of the heart by catecholamines might be an important reason underlying embryonic death [42]. Feeding pregnant females with dihydroxyphenylserine, a precursor that can be converted to norepinephrine in the absence of DβH, prevented the lethal phenotype and the adult DβH-null mice can survive in the absence of catecholamines [42,79]. Mice lacking the neuronal norepinephrine metabolizing enzyme, monoamine oxidase, showed increased content of norepinephrine in the brain but cardiac phenotype has not been reported [43].

Cho et al. [79] studied cardiac function in adult DβH-deficient mice treated with dihydroxyphenylserine. LV contractility in these mice was elevated at baseline and during β-agonist stimulated. Although total βAR density remains unchanged, the fraction of high-affinity βAR is significantly greater in DβH knockout than control hearts. This is most likely due to a 50% lower βARK1 activity in this strain [79]. This phenotype provides additional evidence for the profound regulation of myocardial contractility by βARK1.

5.3 Disruption of presynaptic {alpha}2AR
Release of norepinephrine from sympathetic nerve varicosities can be facilitated or inhibited by a variety of receptors localized on the presynaptic neuronal membrane. Of these receptors, activation of {alpha}2AR powerfully inhibits norepinephrine release. The individual role of three subtypes, {alpha}2A, {alpha}2B and {alpha}2C, in mediating the presynaptic inhibition is not clear from pharmacological studies. Recent studies with knockout mice revealed that {alpha}2A and {alpha}2C are the presynaptic {alpha}2AR subtypes that inhibit norepinephrine release [45–47]. In cardiac tissue, norepinephrine release by nerve activation was inhibited by {alpha}2A at higher and by {alpha}2C at lower stimulatory frequencies [47]. Furthermore, {alpha}2AR-lacking mice display tachycardia, higher plasma levels of catecholamines, reduced norepinephrine content and βAR density, and suppressed AC activity upon β-agonist stimulation [47,80,81]. Thus, excessive adrenergic stimulation due to uncontrolled norepinephrine release, down-regulates βAR signaling. Interestingly, duel {alpha}2A/{alpha}2B knockout mice developed ventricular hypertrophy with suppressed LV contractility [47].

5.4 Summary
Presence of neuronally-derived catecholamines is essential for embryonic development but is not a necessity for adulthood survival. On the contrary, excessive sympathetic drive to the heart, due to genetic manipulations, leads to expected phenotypes, including hypertrophic growth, interstitial fibrosis, myocardial dysfunction and βAR desensitization. These mouse strains provide novel models for studies on the changes originated from uncontrolled cardiac sympathetic activation.


   6 Gene complementation
Top
 1 Introduction
 2 Phenotypes of mice...
 3 Phenotype of strains...
 4 Comparison of cardiac...
 5 Presynaptic factors...
 6 Gene complementation
7 Conclusions
 References
 
An important part of research using gene-targeted mice is to select candidate genes suitable for gene therapy of HF. One approach established recently is to overexpress a ‘therapeutic’ gene in the heart of mice with cardiomyopathic phenotype to generate hybrid offspring carrying duel genetic manipulations.

6.1 Gene complementation by crossbreeding
Genetic models of dilated cardiomyopathy and HF used for crossbreed studies include disruption of muscle-LIM protein [82], overexpression of Gq{alpha} [27], expression of a dominant-negative CREB transcription factor, and mutant {alpha}-myosin heavy chain (MHC) or calsequestrin [83–85]. Strains that overexpress βARKct, β2AR and AC were mated with HF strains to introduce therapeutic genes (Table 2).


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  		Table 2 Outcomes of crossbreeding studies to enhance cardiac β-adrenergic activity in strains of mice with cardiomyopathya

 
Rockman et al. [86] crossed MLP-deficient mice with either βARKct- or β2AR-overexpressing lines. MLP-null mice develop a dilated cardiomyopathy, reduction in LV fractional shortening and dP/dtmax, and markedly blunted responses to β-agonist stimulation. Compared to wild-type mice, the content and activity of βARK1 in the MLP knockout mice were doubled and AC activity was suppressed [82,86]. In hybrid mice from MLP-deficient–/βARKct-crossing, all abnormalities in cardiac function and β-adrenergic signaling were reversed [86]. In contrast, 200-fold overexpression of β2AR in MLP-lacking mice reduced survival and facilitated the onset of HF [86].

Using a similar approach, Dorn et al. [87] crossed mice overexpressing Gq{alpha} with that overexpressing βARKct or β2AR at three levels. In contrast to MLP-deficient model, expression of βARKct in Gq{alpha} transgenic mice failed to reverse the development of cardiac hypertrophy and dysfunction and the effect of β2AR overexpression is dose-dependent. Whereas 1000-fold overexpression of β2AR exaggerated cardiac abnormalities and 140-fold overexpression had no beneficial effect, 30-fold β2AR overexpression in Gq{alpha} mice improved LV contractility and fractional shortening, suppressed hypertrophy and normalized AC activity [87]. The contradictory outcomes of cardiac expression of βARKct in MLP-deficient versus Gq{alpha}-overexpressing strains may lay in the fact that the βARK1 activity in Gq{alpha} mice was not elevated [87]. Two groups have successfully rescued the phenotype in Gq{alpha}-overexpressing mice by crossing these mice with strains overexpressing AC [32,88]. A complete prevention of cardiac dysfunction in Gq{alpha}/ACV mice was observed although myocardial hypertrophy and expression of hypertrophy-associated genes remained unaffected [32].

The effect of βARKct expression remains controversy because of findings in another three strains with cardiomyopathy (Table 2). Expression of βARKct in calsequestrin-overexpressing mice reduced βARK1 activity, limited ventricular dilatation and prevented immature mortality [89]. However, this intervention only leads to partial improvement of ventricular function in male mice expressing mutant {alpha}-MHC [90], and shows no effect in the dominant-negative CREB line (a model with normal βARK1 activity) (Koch WJ, personal communication, 1999). Co-expression of β2AR either facilitated the development of HF and reduced survival in the mutant {alpha}-MHC mice [90] or failed to alleviate cardiomyopathy and dysfunction in the dominant-negative CREB mice (Table 2).

6.2 Summary
Crossbreed studies have proven to be a powerful tool in vivo to assess the function of genes with potential for gene therapy of HF. These studies reveal that interventions that upregulate β-adrenergic signaling in the heart are potentially beneficial. Whereas the three lines overexpressing β2AR, βARKct and AC all have enhanced β-adrenergic activity, the therapeutic effect is clearly dependent on the etiology of cardiomyopathy, the type of the ‘rescue’ gene and the level of overexpression (Table 2, Fig. 1). Furthermore, it appears that overexpression of AC and βARK1 is an effective and safe approach for the reversal of HF. For β2AR overexpression, the expression levels should be low to achieve therapeutic effect with no noxious consequences. This notion fits with the beneficial outcomes in in vivo studies showing 5–20-fold β2AR overexpression by adenoviral gene transfection [52], and a facilitated transition from compensatory hypertrophy to HF following surgically-induced pressure overload in mice overexpressing β2AR by 200-fold [91,92].


Figure 1
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  		Fig. 1 Schematic diagram depicting the β-adrenergic signaling cascade (left) and mouse strains targeting this system at various levels (right). Although overexpression of β1AR, β2AR (at high levels) or Gs{alpha} results in cardiomyopathic phenotype (indicated as *), overexpression of AC, βARKct and β2AR (low levels) or disruption of βARK1 or β-arrestin-1 all improved basal and stimulated myocardial function, through augmented β-adrenergic signaling, with no major adverse consequences. Dashed lines represent inhibitory action. βARK1+/– indicates heterozygous.

 

   7 Conclusions
Top
 1 Introduction
 2 Phenotypes of mice...
 3 Phenotype of strains...
 4 Comparison of cardiac...
 5 Presynaptic factors...
 6 Gene complementation
 7 Conclusions
References
 
Gene manipulation targeting the cardiac sympathoadrenergic signaling systems, together with in vivo murine physiological techniques, has lead to increased recognition regarding the role of the sympathoadrenergic signaling in cardiac physiology in health and disease. Whereas studies on mouse models have confirmed data obtained with classical research approaches, such as receptor agonists and antagonists, surprising and interesting phenotypes have emerged from these studies. In addition, these discoveries have established new concepts with potential targets as novel therapeutic strategies for compromised heart. The rapid increase in the numbers of both transgenic strains and studies using these models calls for timely summary and construction of the ‘phenotype database’ to integrate such exploding information into our knowledge. Although relatively little is known at this time, it also must be appreciated that caution is required in exploration and interpretation of phenotypes of gene-targeted mice since cardiac overexpression or disruption of a gene might alter expression of other related genes thereby modifying the genuine phenotype.

Time for primary review 33 days.


   Acknowledgements
 
This work is supported by Australian National Health and Medical Research Council. I thank the useful comments from Drs Anthony Dart and Gavin Lambert.


   References
Top
 1 Introduction
 2 Phenotypes of mice...
 3 Phenotype of strains...
 4 Comparison of cardiac...
 5 Presynaptic factors...
 6 Gene complementation
 7 Conclusions
 References
 

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