Transgenic Manipulation of Myocardial G Protein–Coupled Receptors and Receptor Kinases
- transgenic mice
- G protein–coupled receptor
- G protein–coupled receptor kinase
- β-adrenergic receptor kinase
The ability to maintain and manipulate mouse embryos in vitro, perfected over the last decade, has launched the expanding field of transgenic experimentation. With the successful insertion of foreign genes into the mouse genome, important transgenic models have emerged in several venues of biomedical research. Transgenic mice permit investigation of the consequences of a protein’s overexpression in a particular tissue. In addition, the loss of function of a protein or enzyme can be examined by tissue-targeted overexpression of an inhibitor peptide or a dominant-negative mutant. Elimination of a protein from all tissues can also be achieved by “knockout” technology, where a gene is disrupted by homologous recombination. These approaches are well suited to study the physiological roles of cellular proteins.
Transgenic models geared toward the study of cardiovascular regulation have recently been described and provide powerful tools to study normal and compromised cardiac physiology. Some of these transgenic models address changes in blood pressure and apolipoprotein levels as well as the consequences of overexpression of specific nuclear factors.1 Most recently, transgenic mice have been developed in which sarcolemmal G protein–coupled receptor signaling has been altered; these animals provide new information regarding the role of signal transduction in cardiac function. Manipulation of various components of the myocardial AR system has led to novel agonist-independent approaches to enhance signaling and augment cardiac function. This mini review summarizes these recent transgenic studies, which have provided unique experimental models for the study of receptor signaling in both normal and diseased myocardium.
Probably the most important receptors involved in cardiac regulation are the ARs. These receptors modulate cardiomyocyte function by coupling to and activating G proteins, which give rise to increases in intracellular second messengers like cAMP and diacylglycerol. In the human heart, ARs are critical regulators of function under both normal and diseased conditions. Myocardial β-ARs, for example, mediate increases in heart rate and contractility in response to increases in the neurotransmitter norepinephrine and the adrenal medullary hormone epinephrine. These two catecholamines bind selectively to ARs, most importantly to β-ARs, present on the sarcolemma.
β-ARs in cardiac muscle include both the β1 and β2 subtypes. In the human heart, as with most mammals, the β1-AR is the predominant subtype, approaching 75% to 80% of total β-ARs.2 Classically, β1- and β2-ARs selectively couple to the adenylyl cyclase stimulatory G protein, Gs, which triggers the catalysis of cAMP formation. Subsequently, this leads to the activation of cAMP-dependent protein kinase (protein kinase A), which targets and phosphorylates several myocardial proteins involved in the positive chronotropic and inotropic response, such as L-type voltage-dependent calcium channels and the sarcoplasmic reticulum protein phospholamban. Activation of both β1- and β2-ARs can lead to increased cardiac contractility2 ; however, recent evidence has demonstrated that β2-ARs can elicit signaling mechanisms that are qualitatively different from those of β1-ARs within cardiac myocytes.3 This cAMP-independent β2-AR regulation of cardiac contractile function has been observed in several species, including humans.3 4 The nature and significance of β2-AR function in the myocardium is not well understood. Notably, signaling through the β-AR–Gs–adenylyl cyclase system undergoes important changes with cardiac disease.2
In addition to β-ARs, cardiac muscle also contains α1-ARs, which are expressed at approximately the same level as β2-ARs.5 Myocytes and myocardial tissue from several species contain both α1A- and α1B-AR subtypes, with the predominant subtype varying among mammals.6 The general paradigm of α1-ARs is that they couple to the G protein, Gq, which stimulates PLC-β, producing the second messengers inositol tris-phosphate and diacylglycerol. These, in turn, increase intracellular calcium concentrations and activate PKC. The exact role of the α1-AR–Gq–PLC-β pathway in myocardial beat-to-beat regulation is not clear, and the precise function of these receptors is unknown.5 6 In fact, it appears that not all α1-ARs in the heart are coupled to this Ca2+-mobilization pathway, since other actions of α1-agonists have been demonstrated in cardiac myocytes, including changes in ionic conductances and pHi, indicating coupling to other G proteins.6 Recent in vitro studies, however, suggest that α1-ARs and other Gq-coupled receptors, which can activate PKC, play a critical role in the initiation of myocyte hypertrophy.7 In addition, stimulation of α1-ARs has been implicated in the preconditioning of myocardium to ischemic injury.8
The regulation of myocardial α- and β-ARs, like that of most G protein–coupled receptors, involves a desensitization mechanism characterized by a rapid loss of receptor responsiveness despite the continued presence of agonist. The desensitization process has been best characterized using the β2-AR system9 and can be initiated by phosphorylation of the agonist-occupied receptor by a serine/threonine kinase known as βARK. βARK1 and a highly homologous isozyme βARK2 are two of the most studied members of the GRK family, which currently consists of six members.10 Desensitization of G protein–coupled receptors requires not only GRK-mediated phosphorylation but also the binding of a second class of proteins, the β-arrestins (β-arrestin-1 and β-arrestin-2), which bind to phosphorylated receptors and sterically interdict further activation of G proteins.9
Regulation of GRKs in the Heart
The GRKs shown to be expressed in the heart are βARK1, βARK2, GRK5, and GRK6, with βARK1 being the most abundant. Since the β1-AR is the most critical receptor for mediating acute changes in myocardial rate and contractility, it is important to note that three of these GRKs (βARK1, βARK2, and GRK5) have been shown to phosphorylate and desensitize β1-ARs in vitro.11
Unique mechanisms for the cellular regulation of βARK1 and other GRKs have recently been elucidated.10 Like most GRKs, βARK1 is a cytosolic enzyme that must translocate to the membrane in order to phosphorylate the activated receptor substrate. The mechanism for translocation of βARK1 (and βARK2) involves the physical interaction between the kinase and the membrane-bound βγ subunits of G proteins (Gβγ). Gβγ, anchored to the membrane through a lipid modification on the carboxyl terminus of the γ subunit (prenylation), is available to interact with βARK after G-protein activation and dissociation. The region of βARK responsible for binding Gβγ has been mapped to a 125–amino acid domain located within the carboxyl terminus of the enzyme.12 Recently, peptides derived from the Gβγ-binding domain of βARK have been shown to act as in vitro βARK inhibitors by competing for Gβγ and preventing translocation.12 13
ARs and GRKs in Heart Disease
Since stimulation of β-ARs is a major mechanism for enhancing myocardial function, this receptor system is particularly critical during cardiac pathophysiology. Changes in β-AR signaling occur in several cardiac disorders, including acute myocardial ischemia, cardiomyopathies, and cardiac transplantation.2 The most well-characterized β-AR–signaling alteration, however, occurs in chronic congestive heart failure, where there is a loss of β-AR density, apparently limited to the β1-AR subtype.14 Both β1-AR mRNA and protein are reduced by ≈50%, which results in a higher percentage of β2-ARs. High levels of catecholamines during heart failure may also serve to desensitize the remaining β-ARs, probably through GRK-mediated mechanisms. In fact, βARK1 levels have been shown to be markedly elevated in tissue samples taken from the LV of failing human hearts.15 This is consistent with, and may contribute to, the functional uncoupling of cardiac β-ARs seen in this condition. Myocardial levels of βARK2 and both β-arrestin isoforms appear to be unchanged in human heart failure,15 whereas levels of GRK5 have yet to be studied. β-AR desensitization also appears to be critical in a more acute setting, as demonstrated by the documented loss of β-AR responsiveness and functional uncoupling following cardiopulmonary bypass.16
ARs may also play a role in myocardial hypertrophy. Most cardiac diseases manifest some degree of hypertrophy, which represents an initial compensatory state; frequently, these conditions ultimately progress to heart failure. The biochemical mediators of hypertrophy are poorly understood, but stimulation of α1-ARs (and other Gq-coupled receptors) in vivo can initiate cardiomyocyte hypertrophy. Thus, the Gq-PLC-PKC pathway may be important in the hypertrophy associated with cardiac diseases.
Transgenic Manipulation of Myocardial ARs
Transgenic technology coupled with the availability of cardiac-specific promoters, like those derived from the genes for ANF, myosin light-chain-2, and α-MHC, has recently made it possible to target AR-signaling alterations specifically to the myocardium. Creation and study of transgenic animals overexpressing different components of myocardial G protein–coupled receptor systems have increased our understanding of the role of receptors, such as β-ARs, in cardiac physiology. Manipulating receptor function genetically may also represent a novel therapeutic approach to improving cardiac function.
The first reported attempt at altering myocardial receptor levels was the generation of transgenic mice with atrial-specific overexpression of the β1-AR.17 This was accomplished by using the human ANF promoter, which produced mice with 5- to 10-fold overexpression of β1-ARs in atrial membranes.17 This moderate overexpression, limited to the atria, was accompanied by minimal effects on signaling.
Using the human β2-AR linked to the murine α-MHC promoter,18 our laboratory was able to overexpress β2-ARs in all chambers of the mouse heart.19 The α-MHC promoter is active mainly in the atria during embryonic development but becomes active in the ventricles at birth, as the α-MHC is the predominant heavy chain isoform in the adult mouse heart. α-MHC–targeted β2-AR transgene expression was unexpectedly robust with >100-fold overexpression in two separate lines of mice.19 In the highest expressing line, β-AR density in the heart approached 40 pmol/mg membrane protein, which is as high or higher than the values reached in cell culture. Surprisingly, β-AR signaling in the hearts of these animals was maximal even in the absence of exogenous agonist, as assessed by the measurement of several biochemical and physiological parameters. Baseline membrane adenylyl cyclase activity from transgenic hearts was increased twofold over baseline control (nontransgenic) activity and equaled maximal agonist-stimulated control activity. Consistent with this biochemical phenotype, isometric tension studies using isolated atria from these animals demonstrated maximal tension in the absence of agonist.19
Paralleling cyclase and isometric tension data, in vivo hemodynamic measurements of these β2-AR transgenic mice revealed maximum cardiac contractility, as measured by LV dP/dtmax, under baseline conditions.19 These β2-AR mice have also recently been shown to have markedly enhanced myocardial relaxation accompanied by downregulation of the sarcoplasmic reticulum protein phospholamban.20 This is consistent with a recent report of enhanced contractility and relaxation in mice in which the phospholamban gene was ablated.21 Contractility in these mice, like that in the β2-AR–overexpressing mice, was not further stimulated by an exogenous β-agonist.
Myocardial receptors are hypothesized to exist as an equilibrium between R, the predominant inactive conformation, and R*, an activated conformation that couples to G proteins. The presence of an agonist, which binds to R*, affects a shift toward this active conformation (panel A of the Figure⇓). The maximum biochemical and physiological effects seen in the β2-AR–overexpressing transgenic animals are likely due to the significant increase in spontaneously isomerized receptors present in the active conformation, in the absence of agonist. With 200-fold overexpression, even the minor fraction of receptors thought to be naturally undergoing this agonist-independent conformational change (from R to R*) becomes significant and results in activation of adenylyl cyclase and the physiological response.19 22 Thus, when wild-type receptors are overexpressed at this extraordinary level, the resultant phenotype seen is what would be expected from expression of mutant ARs, which have been shown to be constitutively active. These constitutively activated mutant ARs, which have been described in detail,23 result from point mutations within the third intracellular domain of the receptor protein. These mutations apparently alleviate certain structural constraints, resulting in a much greater proportion of receptors spontaneously in the R* conformation. Thus, two different mechanisms account for the activated phenotype seen with overexpression of wild-type versus mutated constitutively active receptors. For the wild-type receptors, the increase in R* is a consequence of the sheer overexpression of the receptors (panel B of the Figure⇓), whereas for the constitutively active mutant receptors, it is a consequence of an increase in the fraction of receptors in the R* state (panel D of the Figure⇓).
The increased cellular concentration of the R* conformation present in the β2-AR–overexpressing mice allows for the study of receptor signaling in the absence of an agonist. This model has been used for the further characterization of the class of receptor ligands known as inverse agonists.22 Inverse agonists, unlike agonists that prefer R* (panel A of the Figure⇑), bind preferentially to R, driving the equilibrium further toward R, thus decreasing signaling. Inverse agonists are also distinct from neutral antagonists, which bind with equal affinity to either R or R* and do not perturb the equilibrium. The phenomenon of increased R* that is present in the hearts of β2-AR–overexpressing mice has been demonstrated by the administration of the inverse agonist ICI-118,551, which caused the diminution of all basal biochemical and physiological parameters, including basal contractility.19 22
These mice also demonstrate the potential of genetic engineering as a novel means for enhancing ventricular function. Given the well-characterized deficiencies in the β-AR system in chronic congestive heart failure, in vivo gene transfer of β-ARs may augment receptor function and provide positive inotropy. Such an approach may be successful, since β-agonists are powerful inotropic agents and acutely treat congestive heart failure, whereas their chronic use appears ineffective, probably limited by functional uncoupling or further receptor downregulation. Increasing receptor density may thus represent a novel way of activating signal transduction independent of agonist.
As alluded to above, another potential way to increase the number of receptors in the R* conformation is to transfer constitutively activated mutant receptors to the myocardium. This has been accomplished in mice by the α-MHC–targeted expression of a transgene encoding a constitutively activated mutant of the α1B-AR.24 This mutant receptor has been shown to evoke agonist-independent activation of the Gq-PLC signaling pathway.23 These mice were generated to investigate the role of the α1-AR–Gq pathway in myocardial hypertrophy. It has been hypothesized that this pathway may be involved in pressure-induced or volume overload–induced hypertrophy. However, this has been difficult to test, since administration of α-agonists evokes peripheral vascular effects that secondarily induce myocardial hypertrophy. In mice with cardiac overexpression of this α1B-AR mutant, hearts were significantly larger, and ventricular myocyte size was increased 62%.24 Other properties associated with ventricular hypertrophy were also seen, including increased ventricular ANF expression. These mutant α1-AR mice are in contrast to the β2-AR–overexpressing animals, which featured marked functional changes without myocardial hypertrophy. These mice, overexpressing a constitutively active α1B-AR (for which R* is the predominant conformation), demonstrate that continuous activation of the Gq-coupled pathway can induce ventricular hypertrophy. They provide a model of ventricular hypertrophy independent of hemodynamic changes. Thus, in addition to overexpression of wild-type receptors, constitutively active receptors provide another potential source of R* and mode for enhanced signaling (panel D of the Figure⇑).
Transgenic Manipulation of Receptor Kinases
A potentially novel way to increase signaling, which has only recently become approachable experimentally, is the prevention of desensitization by inhibiting GRK-mediated phosphorylation. With the finding that βARK1 levels are elevated in chronic heart failure,15 which may contribute to the loss of β-AR responsiveness, inhibition of βARK1 becomes a potential target for increasing receptor signaling. We have recently generated transgenic mice with myocardial expression of a peptide inhibitor of βARK1.25 The inhibitor of myocardial βARK is the carboxyl terminus of βARK1, which competes for Gβγ binding to the kinase, a process required for enzyme activation.12 Transgenic mice expressing the βARK inhibitor in their hearts have increased basal in vivo LV contractility and supersensitivity to exogenous isoproterenol.25 These results indicate that βARK may exert a tonic inhibitory effect on myocardial β1-ARs even in the absence of agonist. The cardiac phenotype demonstrated in these mice further supports the hypothesis that isomerization of receptors to R* occurs under basal conditions, since this conformation is the substrate for GRK phosphorylation. In mice with myocardial βARK inhibited, basal R* is presumably less phosphorylated and can proceed to stimulate Gs, producing cAMP and enhancing myocardial function (panel C of the Figure⇑).
Strengthening the hypothesis that myocardial βARK activity is critical to cardiac physiology is the phenotype demonstrated by transgenic mice overexpressing βARK1.25 In mice with threefold to fivefold more βARK1 activity in the myocardium, there was a significant attenuation of isoproterenol-induced inotropy. Myocardial β-ARs (β1 and β2 subtypes) are therefore in vivo substrates for this important GRK. This finding further supports the theory that increased βARK1 may contribute to the attenuation of β-AR signaling in heart failure. Furthermore, βARK1 inhibition may represent a novel therapeutic approach to improve myocardial function in patients with heart failure. Notably, in this regard, β-antagonists, which may be effective chronic therapy for heart failure,26 appear to decrease βARK1 activity.27 This finding, coupled with that of increased βARK1 expression in a state of chronic β-adrenergic stimulation, such as is seen in heart failure, suggests that βARK1 expression may be coupled very tightly to levels of β-AR signaling. We are currently pursuing in vivo gene transfer experiments in adult myocardium to test the effects of βARK inhibition and β-AR overexpression on cardiac function. After successful accomplishment of the difficult task of optimizing global gene delivery to adult myocardium, this approach should provide additional answers concerning the role of β-AR signaling in cardiac pathophysiology as well as the potential use of gene therapy in cardiac disease.
Transgenic manipulation of the myocardial β-AR–Gs–adenylyl cyclase signaling system has not been limited to the receptor or receptor kinase. Transgenic mice with myocardial overexpression of the α subunit of Gs were recently described.28 These mice, which had approximately threefold Gsα overexpression, demonstrated no change in the steady state maximal cyclase activity.28 At a physiological level, as assessed by echocardiography on 10-month-old animals, there was no change in basal contractility, but infusions of catecholamines led to enhanced contractility and increased heart rate relative to control animals.29 However, histological analysis of older (16-month) transgenic mouse hearts revealed some fibrosis, but function was not reported in these older animals. The authors concluded that Gsα overexpression enhanced the efficacy of the β-AR–Gs–adenylyl cyclase signaling pathway, which over the life of the animal led to myocardial damage, thus providing a model of cardiomyopathy.29
Comparison of the phenotype of the Gsα animals with those of the β2-AR and βARK inhibitor animals discussed above, however, suggests that several of the conclusions drawn in these studies may be unwarranted. First, in contrast to the β2-AR and βARK inhibitor animals, the phenotype of the Gsα-overexpressing animals is less pronounced, since neither adenylyl cyclase alterations nor changes in basal contractility could be demonstrated. This calls into question whether β-AR–Gs activation really occurs in this model under physiological circumstances. Second, it is difficult to understand how the model provides insight into cardiomyopathy or heart failure when the only physiological change noted was an increase in function. Third, no evidence has been provided to support the authors’ (Gaudin et al28 and Iwase et al29 ) conclusion that the pathological changes observed have anything to do with long-term β-AR stimulation (eg, their elimination by chronic β-AR blockade) as opposed to some other manifestation of signaling by overexpressed Gsα. For example, the β2-AR–overexpressing animals also develop mild histological changes after very long-term expression (>10 months), but animals overexpressing the βARK inhibitor show no such changes even at 15 months of age, despite the fact that both types of transgenic animals have a much more striking enhancement of cardiac function than do Gsα overexpressors (W.J. Koch, C.A. Milano, and R.J. Lefkowitz, unpublished data, 1995). Therefore, myocyte pathology is by no means an invariant consequence of long-term enhancement of cardiac function by genetic manipulation of the β-AR–Gs–adenylyl cyclase pathway. It also indicates that much more work is necessary to understand the pathological mechanisms involved in each instance.
Finally, the late development of these pathological changes in some of the transgenic models is not really germane to the issue of whether such manipulations might be advantageous to provide short-term inotropic support of the heart (eg, in settings similar to those in which dobutamine infusions are currently used). Of course, in the case of βARK inhibition (pharmaceutical or genetic), the absence of histological changes in the older animals suggests that even long-term therapy might be beneficial.
The availability of cDNA clones encoding ARs and GRKs, coupled with transgenic animal technology and myocardial-specific promoters, has made it possible to study the consequences of specific alterations in the myocardial AR systems in vivo. Studies involving these transgenic mice have advanced the understanding of signaling through ARs in the heart, both under normal and pathophysiological conditions, and transgenic mice represent important experimental models for testing hypotheses about receptor theory, myocardial hypertrophy, and therapeutics. Furthermore, transgenic mice overexpressing β2-ARs and a βARK inhibitor suggest that gene transfer may represent an exciting novel therapeutic approach for cardiovascular disease. These initial studies using transgenic mice should also stimulate additional studies using other myocardial signaling systems, which will, no doubt, yield significant and exciting results.
Selected Abbreviations and Acronyms
|ANF||=||atrial natriuretic factor|
|GRK||=||G protein–coupled receptor kinase|
|LV||=||left ventricle, left ventricular|
|MHC||=||myosin heavy chain|
|PKC||=||protein kinase C|
We wish to thank our collaborators who contributed to our described transgenic studies, especially Drs H.A. Rockman and R.A. Bond. We also wish to thank Dr S. Vatner for making his manuscript29 available before publication and M. Holben and D. Addison for secretarial assistance.
- Received September 27, 1995.
- Accepted January 3, 1996.
- © 1996 American Heart Association, Inc.
Xiao R-P, Lakatta EG. β1-Adrenoceptor stimulation and β2-adrenoceptor stimulation differ in their effects on contraction, cytosolic Ca2+, and Ca2+ current in single rat ventricular cells. Circ Res.. 1993;73:286-300.
Altschuld RA, Starling RC, Hamlin RL, Billman GE, Hensley J, Castillo L, Fertel RH, Hohl CM, Robitaille P-ML, Jones LR, Xiao R-P, Lakatta EG. Response of failing canine and human heart cells to β2-adrenergic stimulation. Circulation. 1995;92:1612-1618.
Bristow MR, Minobe W, Rasmussen R, Hershberger RE, Hoffman BB. Alpha-1 adrenergic receptors in the nonfailing and failing human heart. J Pharmacol Exp Ther.. 1988;247:1039-1045.
Kariya K, Karns LR, Simpson PC. Expression of a constitutively activated mutant of the β-isozyme of protein kinase C in cardiac myocytes stimulates the promoter of the β-myosin heavy chain isogene. J Biol Chem.. 1991;266:10023-10026.
Tsuchida A, Liu Y, Liu GS, Cohen MV, Downey JM. α1-Adrenergic agonists precondition rabbit ischemic myocardium independent of adenosine by direct activation of protein kinase C. Circ Res.. 1994;75:576-585.
Hausdorff WP, Caron MG, Lefkowitz RJ. Turning off the signal: desensitization of β-adrenergic receptor function. FASEB J.. 1990;4:2881-2889.
Inglese J, Freedman NJ, Koch WJ, Lefkowitz RJ. Structure and mechanism of the G protein-coupled receptor kinases. J Biol Chem.. 1993;268:23735-23738.
Freedman NJ, Liggett SB, Drachman DE, Pei G, Caron MG, Lefkowitz RJ. Phosphorylation and desensitization of the human β1-adrenergic receptor: involvement of G protein-coupled receptor kinases and cAMP-dependent protein kinase. J Biol Chem.. 1995;270:17953-17961.
Koch WJ, Inglese J, Stone WC, Lefkowitz RJ. The binding site for the βγ subunits of heterotrimeric G proteins on the β-adrenergic receptor kinase. J Biol Chem.. 1993;268:8256-8260.
Boekhoff I, Inglese J, Schleicher S, Koch WJ, Lefkowitz RJ, Breer H. Olfactory desensitization requires membrane targeting of receptor kinase mediated by βγ-subunits of heterotrimeric G proteins. J Biol Chem.. 1994;269:37-40.
Bristow MR, Minobe WA, Reynolds MV, Port JD, Rasmussen R, Ray PE, Feldman AM. Reduced β1 receptor messenger RNA abundance in the failing human heart. J Clin Invest.. 1993;92:2737-2745.
Lohse MJ. G-protein-coupled receptor kinases and the heart. Trends Cardiovasc Med. 1995;5:63-68.
Schwinn DA, Leone BJ, Spahn DR, Chesnut LC, Page SO, McRae RL, Liggett SB. Desensitization of myocardial β-adrenergic receptors during cardiopulmonary bypass: evidence for early uncoupling and late downregulation. Circulation. 1991;84:2559-2567.
Bertin B, Mansier P, Makeh I, Briand P, Rostene W, Swynghedauw B, Strosberg AD. Specific atrial overexpression of G protein coupled human β1 adrenoceptors in transgenic mice. Cardiovasc Res.. 1993;27:1606-1612.
Subramaniam A, Jones WK, Gulick J, Wert S, Neumann J, Robbins J. Tissue-specific regulation of the α-myosin heavy chain gene promoter in transgenic mice. J Biol Chem.. 1991;266:24613-24620.
Milano CA, Allen LF, Rockman HA, Dolber PC, McMinn TR, Chien KR, Johnson TD, Bond RA, Lefkowitz RJ. Enhanced myocardial function in transgenic mice overexpressing the β2-adrenergic receptor. Science.. 1994;264:582-586.
Rockman HA, Hamilton RA, Milano CA, Bhargava V, Mao L, Lefkowitz RJ. Enhanced myocardial relaxation in vivo in transgenic mice overexpressing the β2-adrenergic receptor. J Am Coll Cardiol.. 1995;25:26A. Abstract.
Luo W, Grupp IL, Harrer J, Ponniah S, Grupp G, Duffy JJ, Doetschman T, Kranias EG. Targeted ablation of the phospholamban gene is associated with markedly enhanced myocardial contractility and loss of β-agonist stimulation. Circ Res. 1994;75:401-409.
Milano CA, Dolber PC, Rockman HA, Bond RA, Venable ME, Allen LF, Lefkowitz RJ. Myocardial expression of a constitutively active α1B-adrenergic receptor in transgenic mice induces cardiac hypertrophy. Proc Natl Acad Sci U S A.. 1994;91:10109-10113.
Koch WJ, Rockman HA, Samama P, Hamilton RA, Bond RA, Milano CA, Lefkowitz RJ. Cardiac function in mice overexpressing the β-adrenergic receptor kinase or βARK inhibitor. Science.. 1995;268:1350-1353.
Bristow MR, O’Connell JB, Gilbert EM, French WJ, Leatherman G, Kantrowitz NE, Orie J, Smucker ML, Marshall G, Kelly P, Deitchman D, Anderson JL, for the bucindolol investigators. Dose-response of chronic β-blocker treatment in heart failure from either idiopathic dilated or ischemic cardiomyopathy. Circulation. 1994;89:1632-1642.
Ping P, Gelzer-Bell R, Roth DA, Kiel D, Insel PA, Hammond HK. Reduced β-adrenergic receptor activation decreases G-protein expression and β-adrenergic receptor kinase activity in porcine heart. J Clin Invest.. 1995;95:1271-1280.
Gaudin C, Ishikawa Y, Wight DC, Mahdavi V, Nadal-Ginard B, Wagner TE, Vatner DE, Homcy CJ. Overexpression of Gsα protein in the hearts of transgenic mice. J Clin Invest. 1995;95:1676-1683.
Iwase M, Bishop SP, Uechi M, Vatner DE, Shannon RP, Kudej RK, Wight DC, Wagner TE, Ishikawa Y, Homcy CJ, Vatner SF. Adverse effects of chronic endogenous sympathetic drive induced by cardiac Gsα overexpression. Circ Res. 1996;78:517-524.