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Low levels of the molecular inotrope S100A1 are sufficient to rescue post-ischemic heart failure (HF). As a prerequisite to clinical application and to determine the safety of myocardial S100A1 DNA-based therapy, we investigated the effects of high myocardial S100A1 expression levels on the cardiac contractile function and occurrence of arrhythmia in a preclinical large animal HF model. At 2 weeks after myocardial infarction domestic pigs presented significant left ventricular (LV) contractile dysfunction. Retrograde application of AAV6-S100A1 (1.5 × 10(13) tvp) via the anterior cardiac vein (ACV) resulted in high-level myocardial S100A1 protein peak expression of up to 95-fold above control. At 14 weeks, pigs with high-level myocardial S100A1 protein overexpression did not show abnormalities in the electrocardiogram. Electrophysiological right ventricular stimulation ruled out an increased susceptibility to monomorphic ventricular arrhythmia. High-level S100A1 protein overexpression in the LV myocardium resulted in a significant increase in LV ejection fraction (LVEF), albeit to a lesser extent than previously reported with low S100A1 protein overexpression. Cardiac remodeling was, however, equally reversed. High myocardial S100A1 protein overexpression neither increases the occurrence of cardiac arrhythmia nor causes detrimental effects on myocardial contractile function in vivo. In contrast, this study demonstrates a broad therapeutic range of S100A1 gene therapy in post-ischemic HF using a preclinical large animal model.

Background and Purpose: We investigated whether β(2) -adrenoceptor overexpression could promote angiogenesis and improve blood perfusion and left ventricular (LV) remodeling of the failing heart., Experimental Approach: We explored the angiogenic effects of β(2) -adrenoceptor overexpression in a rat model of post-myocardial infarction (MI) heart failure (HF). Cardiac adenoviral-mediated β(2) -adrenoceptor overexpression was obtained via direct intramyocardial injection 4-weeks post-MI. Adenovirus(Ad)-GFP and saline injected rats served as controls. Furthermore, we extended our observation to β(2) -adrenoceptor -/- mice undergoing MI., Key Results: Transgenes were robustly expressed in the LV at 2 weeks post-gene therapy, whereas their expression was minimal at 4-weeks post-gene delivery. In HF rats, cardiac β(2) -adrenoceptor overexpression resulted in enhanced basal and isoprenaline-stimulated cardiac contractility at 2-weeks post-gene delivery. At 4 weeks post-gene transfer, Ad-β(2) -adrenoceptor HF rats showed improved LV remodeling and cardiac function. Importantly, β(2) -adrenoceptor overexpression was associated with a markedly increased capillary and arteriolar length density and enhanced in vivo myocardial blood flow and coronary reserve. At the molecular level, cardiac β(2) -adrenoceptor gene transfer induced the activation of the VEGF/PKB/eNOS pro-angiogenic pathway. In β(2) -adrenoceptor-/- mice, we found a ~25% reduction in cardiac capillary density compared with β(2) -adrenoceptor+/+ mice. The lack of β(2) -adrenoceptors was associated with a higher mortality rate at 30 days and LV dilatation, and a worse global cardiac contractility compared with controls., Conclusions and Implication: β(2) -Adrenoceptors play an important role in the regulation of the angiogenic response in HF. The activation of VEGF/PKB/eNOS pathway seems to be strongly involved in this mechanism., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

Heart failure (HF) is a common pathological end point for several cardiac diseases. Despite reasonable achievements in pharmacological, electrophysiological and surgical treatments, prognosis for chronic HF remains poor. Modern therapies are generally symptom oriented and do not currently address specific intracellular molecular signaling abnormalities. Therefore, new and innovative therapeutic approaches are warranted and, ideally, these could at least complement established therapeutic options if not replace them. Gene therapy has potential to serve in this regard in HF as vectors can be directed toward diseased myocytes and directly target intracellular signaling abnormalities. Within this review, we will dissect the adrenergic system contributing to HF development and progression with special emphasis on G-protein-coupled receptor kinase 2 (GRK2). The levels and activity of GRK2 are increased in HF and we and others have demonstrated that this kinase is a major molecular culprit in HF. We will cover the evidence supporting gene therapy directed against myocardial as well as adrenal GRK2 to improve the function and structure of the failing heart and how these strategies may offer complementary and synergistic effects with the existing HF mainstay therapy of β-adrenergic receptor antagonism.

Heart failure (HF) is the common end point of cardiac diseases. Despite the optimization of therapeutic strategies and the consequent overall reduction in HF-related mortality, the key underlying intracellular signal transduction abnormalities have not been addressed directly. In this regard, the gaps in modern HF therapy include derangement of β-adrenergic receptor (β-AR) signaling, Ca(2+) disbalances, cardiac myocyte death, diastolic dysfunction, and monogenetic cardiomyopathies. In this review we discuss the potential of gene therapy to fill these gaps and rectify abnormalities in intracellular signaling. We also examine current vector technology and currently available vector-delivery strategies, and we delineate promising gene therapy structures. Finally, we analyze potential limitations related to the transfer of successful preclinical gene therapy approaches to HF treatment in the clinic, as well as impending strategies aimed at overcoming these limitations.

We use a novel technique that allows for closed recirculation of vector genomes in the cardiac circulation using cardiopulmonary bypass, referred to here as molecular cardiac surgery with recirculating delivery (MCARD). We demonstrate that this platform technology is highly efficient in isolating the heart from the systemic circulation in vivo. Using MCARD, we compare the relative efficacy of single-stranded (ss) adeno-associated virus (AAV)6, ssAAV9 and self-complimentary (sc)AAV6-encoding enhanced green fluorescent protein, driven by the constitutive cytomegalovirus promoter to transduce the ovine myocardium in situ. MCARD allows for the unprecedented delivery of up to 48 green fluorescent protein genome copies per cell globally in the sheep left ventricular (LV) myocardium. We demonstrate that scAAV6-mediated MCARD delivery results in global, cardiac-specific LV gene expression in the ovine heart and provides for considerably more robust and cardiac-specific gene delivery than other available delivery techniques such as intramuscular injection or intracoronary injection; thus, representing a potential, clinically translatable platform for heart failure gene therapy.

Driven, at least in part, by the National Institutes of Health roadmap, an increasing number of studies has bridged the chasm between observations in the basic research laboratory and the clinical bedside. These studies have been an integral part in "translating" new discoveries into therapeutic initiatives. However, "translational medicine" has been used less frequently in the development of cardiovascular drugs or in predicting the potential cardiovascular toxicity of non-cardiac agents. Studies in animal models can provide important clues as to the potential cardiotoxicity of new therapeutic agents, as well as providing a template for the rational design of clinical trials. Three examples of drug development programs that might have been altered by clues available from laboratory studies include the development programs for the anti-cancer drug trastuzumab, the cyclooxygenase inhibitors, and the adenosine-receptor agonists and antagonists. Although mouse models may not always represent the physiology of humans, they provide important information that clinical scientists can utilize in designing safe programs for the evaluation of new pharmacologic agents.

S100A1, a Ca superset2+-binding protein of the EF-hand type, is preferentially expressed in myocardial tissue and has been shown to enhance cardiac contractile performance by regulating both sarcoplasmic reticulum (SR) Ca superset2+-handling and myofibrillar Ca superset2+-responsiveness. In cardiac disease, the expression of S100A1 is dynamically altered as it is significantly down-regulated in end stage human heart failure (HF), and it is up-regulated in compensated hypertrophy. Therefore, the delivery of a transgene encoding for S100A1 to the myocardium might be an attractive strategy for improving cardiac function in HF by replacing lost endogenous S100A1. In this study we sought to test whether exogenous S100A1 gene delivery to alter global cardiac function is feasible in the normal rabbit heart. An adenoviral S100A1 transgene (AdvS100A1) also containing the green fluorescent protein (GFP) was delivered using an intracoronary injection method with a dose of 5 x 10 superset11 total virus particles (tvp) (n = 8). Rabbits treated with either a GFP-only adenovirus (AdvGFP) or saline were used as control groups (n = 11 each). Seven days after global myocardial in vivo gene delivery hemodynamic parameters were assessed. S100A1 overexpression as a result of the intracoronary delivery of AdvS100A1 significantly increased left ventricular (LV) +dP/dt subsetmax, -dP/dt subsetmin and systolic ejection pressure (SEP) compared to both control groups after administration of isoproterenol (0.1, 0.5 and 1.0 microg/kgBW/min), while contractile parameters remained unchanged under basal conditions. These results demonstrate that global myocardial in vivo gene delivery is possible and that myocardial S100A1 overexpression can increase cardiac performance. Therefore, substitution of down-regulated S100A1 protein expression levels may represent a potential therapeutic strategy for improving the cardiac performance of the failing heart.

The effect of S100A1 protein on cardiac excitation-contraction (E-C) coupling was studied using recombinant human S100A1 protein (0.01-10 microM) introduced into single rabbit ventricular cardiomyocytes via a patch pipette. Voltage clamp experiments (20 degrees C) indicated that 0.1 microM S100A1 increased Ca(2+) transient amplitude by approximately 41% but higher or lower S100A1 concentrations had no significant effect. L-type Ca(2+) current amplitude or Ca(2+) efflux rates via the Na(+)/Ca(2+) exchanger (NCX) were unaffected. The rate of Ca(2+) uptake associated with the SR Ca(2+)-ATPase (SERCA2a) was increased by approximately 22% with 0.1 microM S100A1, but not at other S100A1 concentrations. Based on the intracellular Ca(2+) and I(NCX) signals in response to 10 mM caffeine, no significant change in SR Ca(2+) content was observed with S100A1 (0.01-10 microM). Therefore, 0.1 microM S100A1 appeared to increase the fractional Ca(2+) release from the SR. This result was confirmed by measurements of Ca(2+) transient amplitude at a range of SR Ca(2+) contents. The hyperbolic relationship between these two parameters was shifted to the left by 0.1 microM S100A1. [(3)H]-ryanodine binding studies indicated that S100A1 increased ryanodine receptor (RyR) activity at 0.1 and 0.3 microM Ca(2). As with the effects on E-C coupling, 0.1 microM S100A1 produced the largest effect. Co-immunoprecipitation studies on a range of Ca(2+)-handling proteins support the selective interaction of S100A1 on SERCA2a and RyR. In summary, S100A1 had a stimulatory action on RyR2 and SERCA2a in rabbit cardiomyocytes. Under the conditions of this study, the net effect of this dual action is to enhance the Ca(2+) transient amplitude without significantly affecting the SR Ca(2+) content.

Heart failure represents the endpoint to many triggering cardiovascular pathologies. However, there are molecular and biochemical features that remain common to the failing heart, despite the varying etiologies. Principal among these is heightened activation of the sympathetic nervous system and associated enhancement of adrenergic signaling pathways via the catecholamines, norepinephrine and epinephrine. During heart failure, several hallmark alterations in the adrenergic system contribute to loss of cardiac function. To specifically study these changes in a physiologically relevant setting, we and others have utilized advances in genetically engineered mouse technology. This chapter will discuss the many transgenic and knockout mouse models that have been developed to study the adrenergic system in the normal and failing heart. These models include genetically manipulated alterations of adrenergic receptors, linked heterotrimeric G proteins, and the regulatory G protein-coupled receptor kinases (GRKs). Among the more-interesting information gained from these models is the finding that inhibition of a particular GRK - GRK2 or beta adrenergic receptor kinase 1 (betaARK1) - is a potential novel therapeutic strategy to improve function in the setting of heart failure. Furthermore, we will discuss recent transgenic research that proposes an important role for hypertension in the development of heart failure. Overall, genetically engineered mouse models pertaining to this critical myocardial signaling system have provided novel insight into heart function under normal conditions and during states of dysfunction and failure.

Purpose: Vascular access polytetrafluoroethylene (PTFE) graft failure is a major cause of morbidity in the hemodialysis population. The most common cause of graft failure is thrombosis secondary to stenosis at the venous outflow tract. Venous outflow stenosis is characterized by intimal-medial hyperplasia. We have developed a porcine arteriovenous (AV) graft model that may be used to investigate this proliferative response and aid in the development of new therapies to prevent intimal-medial hyperplasia and improve graft patency., Methods: Left carotid to right external jugular vein PTFE (6 mm) grafts were implanted in the necks of swine. Immediately following anatomosis, flow rates were recorded. In one group of animals (n = 4) the venous outflow tract was harvested after 7 days and morphometric analysis of intimal and medial area was performed. In a second group (n = 8) the graft patency was monitored until 28 days., Results: All porcine PTFE fistula grafts were patent at 7 days and 100% patency was maintained until 14 days. After 28 days, 75% of the grafts failed due to thrombosis. The venous outflow tract developed a significant proliferative response. After 7 days the intimal and medial areas were 469 +/- 9 microm2 and 875 +/- 26 microm2 respectively. At 28 days the intimal and medial areas were 913 +/- 55 microm2 and 1437 +/- 182 microm2 respectively. Luminal flow rate of the venous outflow tract was reduced significantly (344 +/- 11 ml/min at Day 0 to 129 +/- 14 ml/min at Day 7, p < 0.05)., Conclusions: This porcine model rapidly, reliably and robustly reproduces the flow reducing stenosis and intimal-medial hyperplasia at the venous outflow tract of PTFE arteriovenous fistula. It represents a promising tool for investigating the mechanisms of intimal-medial hyperplasia, evaluating therapeutic interventions and new graft materials.

Objective: Adenoviral gene transfer to the arrested heart during cardiopulmonary bypass (CPB) is a novel method of allowing prolonged vector contact with the myocardium. In this model we investigated the importance of temperature, duration of arrest and cardioplegia on transgene expression., Methods: First-generation adenoviral vector (1 x 10(12) total viral particles) containing the transgene for the human beta2-adrenoceptor (Adeno-beta(2)AR) or beta-galactosidase (Adeno-beta(gal)) was delivered to neonatal piglets via the proximal aorta, during simulated cardiac surgery, and allowed to dwell for the cross-clamp duration. Four treatment groups received Adeno-beta(2)AR. Groups A (n=4) and B (n=6) underwent cold crystalloid cardioplegia arrest for 10 and 30 min, respectively, Group C (n=5) underwent warm crystalloid cardioplegia arrest for 10 min, and Group D (n=5) underwent warm fibrillatory arrest for 10 min. Group E (n=6) received Adeno-beta(gal) and underwent cold crystalloid cardioplegia arrest (30 min). Animals were weaned off CPB and recovered for 2 days. Receptor density was assessed in membrane fractions using radioligand binding and compared using the Mann-Whitney U-test., Results: Left ventricular transgene overexpression, as evidenced by elevated betaAR density, following Adeno-beta(2)AR treatment was greatest with cold cardioplegia (Group A 588+/-288.8 fmol/mg; P=0.002 and Group B 520+/-250.9 fmol/mg; P=0.01) versus control (Group E 109+/-8.4 fmol/mg). Overexpression also occurred with warm cardioplegia (Group C 274+/-69.5 fmol/mg; P=0.05) and ventricular fibrillation (Group D 215+/-48.4 fmol/mg; P=0.02) versus control. Comparison of the combined cold cardioplegia groups versus those treated with warm conditions showed a trend towards increased expression with cold conditions (P=0.1). Receptor density was also significantly increased in the right ventricle of animals in Group B (165+/-18.1 fmol/mg; P=0.03) and Group D (181+/-23.4 fmol/mg; P=0.02) versus control (Group E 118+/-5.8 fmol/mg)., Conclusions: Cold crystalloid cardioplegia is not detrimental to gene transfer in vivo. In fact, there was a trend towards increased left ventricular transgene expression when the adenoviral vector was delivered following cold versus warm cardioplegia. Shorter periods of contact with the vector may reduce transgene overexpression. Therefore, gene transfer is possible during cardiac surgery with clinically used myocardial protection techniques.

Molecular changes that take place during the evolution of heart failure (HF), especially the well characterized beta-adrenergic receptor (betaAR) signaling abnormalities, represent attractive targets for myocardial gene therapy. The beta-adrenergic receptor kinase (betaARK1 or GRK2) is a cytosolic enzyme that phosphorylates only agonist-occupied betaARs as well as other G protein-coupled receptors (GPCRs), leading to desensitization and functional uncoupling. betaARK1 levels and activity are elevated in the failing heart and therefore, it has recently been evaluated as a potential target for novel HF treatment. This review summarizes recent results obtained in transgenic mouse models as well as in animals where a betaARK1 inhibitor peptide (betaARKct) was delivered via the coronary arteries by exogenous gene transfer. These results strongly suggest that betaARK1 inhibition may represent a significant improvement in HF therapy.

S100A1, a Ca(2+) binding protein of the EF-hand type, is preferentially expressed in myocardial tissue and has been found to colocalize with the sarcoplasmic reticulum (SR) and the contractile filaments in cardiac tissue. Because S100A1 is known to modulate SR Ca(2+) handling in skeletal muscle, we sought to investigate the specific role of S100A1 in the regulation of myocardial contractility. To address this issue, we investigated contractile properties of adult cardiomyocytes as well as of engineered heart tissue after S100A1 adenoviral gene transfer. S100A1 gene transfer resulted in a significant increase of unloaded shortening and isometric contraction in isolated cardiomyocytes and engineered heart tissues, respectively. Analysis of intracellular Ca(2+) cycling in S100A1-overexpressing cardiomyocytes revealed a significant increase in cytosolic Ca(2+) transients, whereas in functional studies on saponin-permeabilized adult cardiomyocytes, the addition of S100A1 protein significantly enhanced SR Ca(2+) uptake. Moreover, in Triton-skinned ventricular trabeculae, S100A1 protein significantly decreased myofibrillar Ca(2+) sensitivity ([EC(50%)]) and Ca(2+) cooperativity, whereas maximal isometric force remained unchanged. Our data suggest that S100A1 effects are cAMP independent because cellular cAMP levels and protein kinase A-dependent phosphorylation of phospholamban were not altered, and carbachol failed to suppress S100A1 actions. These results show that S100A1 overexpression enhances cardiac contractile performance and establish the concept of S100A1 as a regulator of myocardial contractility. S100A1 thus improves cardiac contractile performance both by regulating SR Ca(2+) handling and myofibrillar Ca(2+) responsiveness.

Background: Increased right ventricular (RV) afterload results in RV hypertrophy and dysfunction, as well as increased levels of intracellular beta-adrenergic receptor kinase (betaARK1). We hypothesize that gene transfer of a betaARK1 inhibitor (betaARKct) may improve RV performance, morbidity, and mortality early after pulmonary artery (PA) banding., Methods: Rabbits underwent PA banding 3 days after right coronary artery injection of an adenovirus containing the gene encoding the betaARKct peptide (n = 14), beta-galactosidase (n = 10), or an empty adenovirus (n = 19). After banding, hemodynamic instability and maximal rate of increase in right ventricular pressure (RV dP/dt(max)) were documented. For 7 days after banding, animals were monitored for mortality, activity, and appetite., Results: When compared with controls, animals receiving the betaARKct transgene showed improvement in survival at 7 days (92.8% +/- 7% vs 48.3% +/- 9%, p = 0.01), less lethargy, a trend toward greater RV dP/dt(max) (NS), and increased hemodynamic stability at the time of banding (78% vs 41%, p = 0.03)., Conclusions: Selective RV expression of betaARKct improves survival and morbidity after PA banding. This represents a novel therapeutic modality for clinical situations involving increased RV afterload.

Background: Acute cardiac contractile dysfunction is common after cardiopulmonary bypass (CPB). A potential molecular mechanism is enhanced beta-adrenergic receptor kinase (betaARK1) activity, because beta-adrenergic receptor (betaAR) signaling is altered in cardiomyocytes after cardioplegia. Therefore, we examined whether adenovirus-mediated intracoronary delivery of a betaARK1 inhibitor (Adv-betaARKct) could prevent post-CPB dysfunction., Methods and Results: Rabbits were randomized to receive 5x10(11) total viral particles of Adv-betaARKct or PBS. After 5 days, hearts were arrested with University of Wisconsin solution, excised, and stored at 4 degrees C for 15 minutes or 4 hours before reperfusion on a Langendorff apparatus. Left ventricular (LV) function measured by end-diastolic pressure response to preload augmentation, contractility (LV dP/dt(max)), and relaxation (LV dP/dt(min)) was assessed by use of increasing doses of isoproterenol and compared with a control group of nonarrested hearts acutely perfused on the Langendorff apparatus. In the PBS-treated hearts, LV function decreased in a temporal manner and was significantly impaired compared with control hearts after 4 hours of cardioplegic arrest. LV function in Adv-betaARKct-treated hearts, however, was significantly enhanced compared with PBS treatment and was similar to control nonarrested hearts even after 4 hours of cardioplegia. Biochemically, several aspects of betaAR signaling were dysfunctional in PBS-treated hearts, whereas they were normalized in betaARKct-overexpressing hearts., Conclusions: Myocardial gene transfer of Adv-betaARKct stabilizes betaAR signaling and prevents LV dysfunction induced by prolonged cardioplegic arrest. Thus, betaARK1 inhibition may represent a novel target in limiting depressed ventricular function after CPB.

An accumulation of recent data on genetically engineered mouse models suggests that results from studies done in vitro are not necessarily duplicated in vivo. The genetic manipulation of the adrenergic receptor (AR) signaling system in the heart has afforded us the opportunity to not only study the physiological impact of AR signaling manipulation but also to examine how the various components interact with one another in vivo. In particular, although members of the G protein-coupled receptor kinase family do not exhibit substrate selectivity when overexpressed in cell culture, in vivo selectivity is apparent when examined in the cardiovascular system of genetically engineered mice. Additionally, transgenic expression of peptide inhibitors of signaling represents a powerful tool to examine specific targets in order to determine their contribution to a physiologic phenotype following stimulation. Finally, in vivo manipulation of the AR system has provided a broader understanding of the role that various G protein-coupled receptors play in situations where multiple members contribute to a phenotype. Thus, although in vitro studies allow for a more defined environment in which to study the signaling mediated by various receptors, it is essential to verify these findings in vivo to confirm or refute in vitro results.

Heart failure (HF) represents one of the leading causes for hospitalization in developed nations. Despite advances in the management of coronary artery disease, no significant improvements in prognosis have been achieved for HF over the last several decades. Heart failure itself represents a final common endpoint for several disease entities, including hypertension, coronary artery disease, and cardiomyopathy. However, certain biochemical features remain common to the failing myocardium. Foremost amongst these are alterations in the beta-adrenergic receptor signalling cascade. Recent advances in transgenic and gene therapy techniques have presented novel therapeutic strategies for the management of HF via enhancement of beta-adrenergic signalling. In this review, we will discuss the biochemical changes that accompany HF as well as corresponding therapeutic strategies. We will then review the evidence from transgenic mouse work supporting the use of adrenergic receptor augmentation in the failing heart and more recent in vivo applications of gene therapy directed at reversing or preventing HF.

Objectives: Using a transgenic mouse model of myocardial-targeted overexpression of the wild-type alpha1B adrenergic receptor (AR) (Tg alpha43), we studied the role of the betaAR kinase (betaARK1) in the evolution of myocardial hypertrophy and its transition to heart failure (HF)., Background: Increased myocardial expression of betaARK1 has been shown to be associated with HF and certain models of hypertrophy., Methods: Tg alpha43 mice and their nontransgenic littermate controls were treated with the alpha1AR agonist phenylephrine (PE) for 3, 7 or 14 days to characterize the cardiac consequences., Results: Nontransgenic littermate control mice treated for 14 days with PE display cardiac hypertrophy with no increase in betaARK1 expression. However, Tg alpha43 animals show a reduced tolerance to 14-day PE treatment, demonstrated by reduced survival and severe cardiac hypertrophy. Moreover, PE treatment for three and seven days in Tg alpha43 mice resulted in an exaggerated hypertrophic response accompanied by significant cardiac biochemical abnormalities that are normally associated with HF, including fetal gene expression, reduced betaAR density and enhanced betaARK1 expression. We also found reduced myocardial stores of the sympathetic neurotransmitter neuropeptide Y., Conclusions: These data suggest that PE-treated Tg alpha43 mice have chronic activation of the cardiac sympathetic nervous system, which may be responsible for the appearance of apparent maladaptive hypertrophy with an evolution towards HF and sudden death. Thus, the cardiac phenotypes found in these mice are not the direct result of enhanced alpha1B AR signaling and suggest that betaARK1 is a key molecule in the transition of myocardial hypertrophy to HF.

The beta-adrenergic receptor (betaAR) kinase (betaARK1) is a G protein-coupled receptor kinase (GRK) that controls cardiac betaAR signaling via receptor phosphorylation, leading to desensitization. We have observed in mice that chronic isoproterenol administration results in increased myocardial levels of betaARK1 activity, suggesting that adrenergic activation can regulate cardiac betaARK1 expression. Thus, we evaluated left ventricular (LV) betaARK1 levels and activity in response to 3 weeks of a low-sodium (0.05%) diet, which is known to chronically activate the sympathetic nervous system. Wistar-Kyoto rats were subjected to either low or regular sodium (2%) intake. To prove the association of betaARK1 expression and low sodium-induced adrenergic activation, a group of rats was subjected to atenolol treatment (1 mg/kg per day) during the low-sodium diet. LV betaARK1 expression was assessed by protein immunoblotting and betaARK1 activity by in vitro GRK phosphorylation assays. We verified the LV protein levels of GRK5, which is abundantly expressed in the heart. A low-sodium diet reduced body weight and cardiac size so that the heart-to-body weight ratio did not change. On the contrary, low-sodium diet increased by 50% both LV betaARK1 protein (densitometry units: normal sodium, 26.5+/-0.9; low sodium, 35.7+/-1.6; P<0.05) and activity (fmol/mg per minute: normal sodium, 6.49+/-1.17; low sodium, 9.15+/-0.93; P<0.05). Atenolol treatment prevented the increase in both protein expression (low sodium plus atenolol, 27.6+/-5.33, P=NS versus normal sodium) and activity (6.54+/-1.19, P=NS versus normal sodium). GRK5 expression was not affected by a low-sodium diet (17.2+/-0.2 versus 18.4+/-0.4, P=NS). Our data indicate that cardiac betaARK1 is regulated by sympathetic action on betaARs as tested by reducing dietary salt and betaAR blockade.

Background: Cardiac gene therapy offers the possibility of enhancing myocardial performance in the compromised heart. However, current gene delivery techniques have limited myocardial transgene expression and pose the risk of extracardiac expression. Isolation of the coronary circulation during cardiac surgery may allow for more efficient and cardiac-selective gene delivery in a clinically relevant model. Methods and Results-- Neonatal piglets (3 kg) underwent a median sternotomy and cardiopulmonary bypass, followed by aortic cross-clamping with 30 minutes of cardioplegic arrest. Adenoviral vectors containing transgenes for either beta-galactosidase (adeno-beta-gal, n=11) or the human beta(2)-adrenergic receptor (adeno-beta(2)-AR, n=15) were administered through the cardioplegia cannula immediately after arrest and were allowed to dwell in the coronary circulation during the cross-clamp period. After 1 week, the animals were killed, and their heart, lungs, and liver were excised and examined for gene expression. Analysis of beta-galactosidase staining revealed transmural myocardial gene expression among animals receiving adeno-beta-gal. No marker gene expression was detected in liver or lung tissue. beta-AR density in the left ventricle after adeno-beta(2)-AR delivery was 396+/-85% of levels in control animals (P<0.01). Animals receiving adeno-beta(2)-AR and control animals demonstrated similar beta-AR density in both the liver (114+/-8% versus 100+/-9%, P=NS) and lung (114+/-7% versus 100+/-9%, P=NS). There was no evidence of cardiac inflammation., Conclusions: By using cardiopulmonary bypass and cardioplegic arrest, intracoronary delivery of adenoviral vectors resulted in efficient myocardial uptake and expression. Undetectable transgene expression in liver or lung tissue suggests cardiac-selective expression.

Road traffic collisions (RTCs) are common precipitants of posttraumatic stress disorder (PTSD). Two preliminary studies suggest that cognitive-behavior therapy (CBT) is, on average, effective in treating this disorder, although the major patterns of treatment outcome remain to be identified. Such outcomes might include treatment response, partial response, and response followed by relapse. To identify these patterns. 50 people with RTC PTSD completed a 12-week course of CBT, with outcome assessment extending to 3-month follow up. Dynamic cluster analyses revealed 2 replicable patterns of outcome: one for responders (n = 30) and one for partial responders (n = 20). Partial responders, compared with responders, tended to have more severe pretreatment numbing symptoms and greater anger about their RTC, along with lower global levels of functioning, greater pain severity and interference, and greater depression and were more likely to be taking psychotropic medications. Responders and partial responders did not differ in homework adherence, number of sessions attended, therapist effects, or stressors occurring during therapy or in the presence or absence of RTC-related litigation. Implications for enhancing treatment outcome are discussed.

Chronic human heart failure is characterized by abnormalities in beta-adrenergic receptor (betaAR) signaling, including increased levels of betaAR kinase 1 (betaARK1), which seems critical to the pathogenesis of the disease. To determine whether inhibition of betaARK1 is sufficient to rescue a model of severe heart failure, we mated transgenic mice overexpressing a peptide inhibitor of betaARK1 (betaARKct) with transgenic mice overexpressing the sarcoplasmic reticulum Ca(2+)-binding protein, calsequestrin (CSQ). CSQ mice have a severe cardiomyopathy and markedly shortened survival (9 +/- 1 weeks). In contrast, CSQ/betaARKct mice exhibited a significant increase in mean survival age (15 +/- 1 weeks; P < 0.0001) and showed less cardiac dilation, and cardiac function was significantly improved (CSQ vs. CSQ/betaARKct, left ventricular end diastolic dimension 5.60 +/- 0.17 mm vs. 4.19 +/- 0.09 mm, P < 0.005; % fractional shortening, 15 +/- 2 vs. 36 +/- 2, P < 0.005). The enhancement of the survival rate in CSQ/betaARKct mice was substantially potentiated by chronic treatment with the betaAR antagonist metoprolol (CSQ/betaARKct nontreated vs. CSQ/betaARKct metoprolol treated, 15 +/- 1 weeks vs. 25 +/- 2 weeks, P < 0.0001). Thus, overexpression of the betaARKct resulted in a marked prolongation in survival and improved cardiac function in a mouse model of severe cardiomyopathy that can be potentiated with beta-blocker therapy. These data demonstrate a significant synergy between an established heart-failure treatment and the strategy of betaARK1 inhibition.

This study examined the effects of cognitive-behavior therapy (CBT) compared with traditional behavior therapy (exposure and response prevention [ERP]) in the group treatment of obsessive-compulsive disorder. Of the 76 participants who started treatment, 38 were wait-listed for 3 months before treatment to assess possible course effects. Both treatments were superior to the control condition in symptom reduction, with ERP being marginally more effective than CBT by end of treatment and again at 3-month follow-up. In terms of clinically significant improvement, treatment groups were equivalent on the conclusion of treatment, but 3 months later significantly more ERP participants met criteria for recovered status. Only 1 of 7 belief measures changed with treatment improvement, and the extent of this cognitive change was similar between CBT and ERP groups. Discussion includes consideration of optimal formats for the delivery of different types of treatment.

Background: Chronic heart failure is associated with impairment of the myocardial beta-adrenergic receptor (beta-AR) system. In this study, the effects of G protein-coupled receptor kinase 5 (GRK5) overexpression on myocardial performance were directly assessed in the hearts of transgenic mice using an isolated work-performing murine heart preparation and computerized analysis of functional data., Methods: A controlled experimental study was performed to evaluate cardiac function in both transgenic mice with a 30-fold overexpression of GRK5 (n = 9, 23 to 29 g) and littermate controls (n = 10, 22 to 29 g). Preload-dependent cardiac output, contractility, stroke work, stroke volume, and heart rate were compared between the two groups., Results: Significant decreases in preload-dependent cardiac output and contractility were observed in the mice with GRK5 overexpression when compared with control group mice and occurred in association with significant decreases in stroke work and stroke volume. There was no significant difference in the average heart rate between the two groups., Conclusions: These data suggest that GRK5 upregulation may be partially responsible for alterations in myocardial function in chronic heart failure.

The medical treatment of chronic heart failure has undergone a dramatic transition in the past decade. Short-term approaches for altering hemodynamics have given way to long-term, reparative strategies, including beta-adrenergic receptor (betaAR) blockade. This was once viewed as counterintuitive, because acute administration causes myocardial depression. Cardiac myocytes from failing hearts show changes in betaAR signaling and excitation-contraction coupling that can impair cardiac contractility, but the role of these abnormalities in the progression of heart failure is controversial. We therefore tested the impact of different manipulations that increase contractility on the progression of cardiac dysfunction in a mouse model of hypertrophic cardiomyopathy. High-level overexpression of the beta(2)AR caused rapidly progressive cardiac failure in this model. In contrast, phospholamban ablation prevented systolic dysfunction and exercise intolerance, but not hypertrophy, in hypertrophic cardiomyopathy mice. Cardiac expression of a peptide inhibitor of the betaAR kinase 1 not only prevented systolic dysfunction and exercise intolerance but also decreased cardiac remodeling and hypertrophic gene expression. These three manipulations of cardiac contractility had distinct effects on disease progression, suggesting that selective modulation of particular aspects of betaAR signaling or excitation-contraction coupling can provide therapeutic benefit.

Background: Understanding the cellular signals that initiate cardiac hypertrophy is of critical importance in identifying the pathways that mediate heart failure. The family of mitogen-activated protein kinases (MAPKs), including the extracellular signal-regulated kinases (ERKs), c-Jun NH(2)-terminal kinase (JNK), and p38 MAPKs, may play specific roles in myocardial growth and function., Methods and Results: To determine the mechanism of activation of MAPK pathways during the development of cardiac hypertrophy, we evaluated the induction of MAPK activity after aortic constriction in wild-type and in 2 types of cardiac gene-targeted mice: one overexpressing a carboxyl-terminal peptide of Galpha(q) that inhibits G(q)-mediated signaling (TG GqI mouse) and another overexpressing a carboxyl-terminal peptide of beta-adrenergic receptor kinase-1 that inhibits Gbetagamma signaling (TG betaARKct mouse). Wild-type mice with pressure overload showed an acute induction of JNK, followed by the induction of p38/p38beta at 3 days and ERK at 7 days. Both JNK and p38 activity remained elevated at 7 days after banding. In TG GqI mice, hypertrophy was significantly attenuated, and induction of ERK and JNK activity was abolished, whereas the induction of p38 and p38beta was robust, but delayed. By contrast, all 3 MAPK pathways were activated by aortic constriction in the TG betaARKct hearts, suggesting a role for Galpha(q), but not Gbetagamma., Conclusions: Taken together, these data show that the induction of ERK and JNK activity in in vivo pressure-overload hypertrophy is mediated through the stimulation of G(q)-coupled receptors and that non-G(q)-mediated pathways are recruited to activate p38 and p38beta.

Background: Genetic manipulation to reverse molecular abnormalities associated with dysfunctional myocardium may provide novel treatment. This study aimed to determine the feasibility and functional consequences of in vivo beta-adrenergic receptor kinase (betaARK1) inhibition in a model of chronic left ventricular (LV) dysfunction after myocardial infarction (MI)., Methods and Results: Rabbits underwent ligation of the left circumflex (LCx) marginal artery and implantation of sonomicrometric crystals. Baseline cardiac physiology was studied 3 weeks after MI; 5x10(11) viral particles of adenovirus was percutaneously delivered through the LCx. Animals received transgenes encoding a peptide inhibitor of betaARK1 (Adeno-betaARKct) or an empty virus (EV) as control. One week after gene delivery, global LV and regional systolic function were measured again to assess gene treatment. Adeno-betaARKct delivery to the failing heart through the LCx resulted in chamber-specific expression of the betaARKct. Baseline in vivo LV systolic performance was improved in Adeno-betaARKct-treated animals compared with their individual pre-gene delivery values and compared with EV-treated rabbits. Total beta-AR density and betaARK1 levels were unchanged between treatment groups; however, beta-AR-stimulated adenylyl cyclase activity in the LV was significantly higher in Adeno-betaARKct-treated rabbits compared with EV-treated animals., Conclusions: In vivo delivery of Adeno-betaARKct is feasible in the infarcted/failing heart by coronary catheterization; expression of betaARKct results in marked reversal of ventricular dysfunction. Thus, inhibition of betaARK1 provides a novel treatment strategy for improving the cardiac performance of the post-MI heart.

We identified abnormalities in the vascular beta-adrenergic receptor (beta-AR) signaling pathway in heart failure after myocardial infarction (MI). To examine these abnormalities, we measured beta-AR-mediated hemodynamics, vascular reactivity, and the vascular beta-AR molecular signaling components in rats with heart failure after MI. Six weeks after MI, these rats had an increased left ventricular (LV) end-diastolic pressure, decreased LV systolic pressure, and decreased rate of LV pressure change (dP/dt). LV dP/dt responses to isoproterenol were shifted downward, although the responses for systemic vascular resistance were shifted upward in heart failure rats (P < 0.05). Isoproterenol- and IBMX-induced vasorelaxations were blunted in heart failure rats (P < 0.05) with no change in the forskolin-mediated vasorelaxation. These changes were associated with the following alterations in beta-AR signaling (P < 0.05): decreases in beta-AR density (aorta: 58.7 +/- 6.0 vs. 35.7 +/- 1.9 fmol/mg membrane protein; carotid: 29.6 +/- 5.6 vs. 18.0 +/- 3.9 fmol/mg membrane protein, n = 5), increases in G protein-coupled receptor kinase activity levels (relative phosphorimage counts of 191 +/- 39 vs. 259 +/- 26 in the aorta and 115 +/- 30 vs. 202 +/- 7 in the carotid artery, n = 5), and decreases in cGMP and cAMP in the carotid artery (0.85 +/- 0.10 vs. 0.31 +/- 0.06 pmol/mg protein and 2.3 +/- 0.3 vs. 1.2 +/- 0.1 pmol/mg protein, n = 5) with no change in Galpha(s) or Galpha(i )in the aorta. Thus in heart failure there are abnormalities in the vascular beta-AR system that are similar to those seen in the myocardium. This suggests a common neurohormonal mechanism and raises the possibility that treatment in heart failure focused on the myocardium may also affect the vasculature.

The goal of this study was to determine whether beta(1)-adrenergic receptor (AR) and beta(2)-AR differ in regulating cardiomyocyte survival and apoptosis and, if so, to explore underlying mechanisms. One potential mechanism is that cardiac beta(2)-AR can activate both G(s) and G(i) proteins, whereas cardiac beta(1)-AR couples only to G(s). To avoid complicated crosstalk between beta-AR subtypes, we expressed beta(1)-AR or beta(2)-AR individually in adult beta(1)/beta(2)-AR double knockout mouse cardiac myocytes by using adenoviral gene transfer. Stimulation of beta(1)-AR, but not beta(2)-AR, markedly induced myocyte apoptosis, as indicated by increased terminal deoxynucleotidyltransferase-mediated UTP end labeling or Hoechst staining positive cells and DNA fragmentation. In contrast, beta(2)-AR (but not beta(1)-AR) stimulation elevated the activity of Akt, a powerful survival signal; this effect was fully abolished by inhibiting G(i), G(beta gamma), or phosphoinositide 3 kinase (PI3K) with pertussis toxin, beta ARK-ct (a peptide inhibitor of G(beta gamma)), or LY294002, respectively. This indicates that beta(2)-AR activates Akt via a G(i)-G(beta gamma)-PI3K pathway. More importantly, inhibition of the G(i)-G(beta gamma)-PI3K-Akt pathway converts beta(2)-AR signaling from survival to apoptotic. Thus, stimulation of a single class of receptors, beta(2)-ARs, elicits concurrent apoptotic and survival signals in cardiac myocytes. The survival effect appears to predominate and is mediated by the G(i)-G(beta gamma)-PI3K-Akt signaling pathway.

Recent improvements in both gene transfer vectors and in vivo gene delivery techniques have facilitated genetic manipulation of myocardial function and enabled targeted therapy of animal models of cardiac disease and, in particular, heart failure. Increases in myocardial perfusion, improved calcium handling, and enhanced beta-adrenergic receptor signaling have all been achieved by gene transfer in animal models, and appear to be important determinants of myocardial function. Increased understanding of the molecular etiologies of myocardial disease processes combined with advances in vectors and gene delivery will facilitate the development of novel therapies and represent important progress in the effort to make myocardial gene therapy a clinical reality beyond experimental protocols.

A mouse model of hypertrophic cardiomyopathy (HCM) was created by expression of a cardiac alpha-myosin transgene including the R(403)Q mutation and a deletion of a segment of the actin-binding domain. HCM mice show early histopathology and hypertrophy, with progressive hypertrophy in females and ventricular dilation in older males. To test the hypothesis that dilated cardiomyopathy (DCM) is part of the pathological spectrum of HCM, we studied chamber morphology, exercise tolerance, hemodynamics, isolated heart function, adrenergic sensitivity, and embryonic gene expression in 8- to 11-mo-old male transgenic animals. Significantly impaired exercise tolerance and both systolic and diastolic dysfunction were seen in vivo. Contraction and relaxation parameters of isolated hearts were also decreased, and lusitropic responsiveness to the beta-adrenergic agonist isoproterenol was modestly reduced. Myocardial levels of the G protein-coupled beta-adrenergic receptor kinase 1 (beta-ARK1) were increased by more than twofold over controls, and total beta-ARK1 activity was also significantly elevated. Induction of fetal gene expression was also observed in transgenic hearts. We conclude that transgenic male animals have undergone cardiac decompensation resulting in a DCM phenotype. This supports the idea that HCM and DCM may be part of a pathological continuum rather than independent diseases.

Background: Transgenic mice with constitutive myocardium-targeted expression of a peptide inhibitor of the ss-adrenergic receptor kinase (ssARKct) have increased in vivo cardiac function and enhanced ss-adrenergic receptor (ssAR) responsiveness., Methods and Results: In the present study, we created transgenic mice with myocardium-targeted ssARKct 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-ssARKct transgenic mice have normal in vivo cardiac contractility and ssAR 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-ssARKct hearts had elevations in left ventricular mass similar to those in NLCs; however, TAC did induce demonstrable ssARKct expression in the transgenic hearts. TAC in NLC mice resulted in an upregulation of myocardial ssARK1 and a loss of ssAR-mediated inotropic reserve. Importantly, although ssARK1 was increased in the hypertrophic CARP-ssARKct mice, the in vivo loss of ssAR responsiveness was not seen after induced ssARKct expression., Conclusions: These results demonstrate that acute ssARK1 inhibition can restore lost myocardial ssAR 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.

The atria and ventricles of transgenic mice (TGbeta(2)) with cardiac overexpression of the human beta(2)-adrenoceptor (beta(2)AR) were initially reported to show maximum contractility in the absence of beta-AR stimulation. However, we have previously observed a different phenotype in these mice, with myocytes showing normal contractility but reduced betaAR responses. We have investigated the roles of cyclic AMP and Gi in basal and betaAR function in these myocytes. ICI 118,551 at inverse agonist concentrations decreased contraction by 32%. However, the cyclic AMP antagonist Rp-cAMPS had no effect on contraction in TGbeta(2) myocytes, indicating that there was no tonic influence of raised cyclic AMP. These findings cannot be explained by the proposed model for inverse agonism, where the activated receptor (R*) raises cyclic AMP levels and so increases contraction in the absence of agonist. After pertussis toxin (PTX) pretreatment to produce inactivation of Gi, the basal contraction in 1 mM Ca(2+) was increased in TGbeta(2) mice (7.82+/-0.47%, n=23) compared to LM mice (3.60+/-0.59%, n=11) (P<0.001). The contraction amplitude of myocytes to the maximal concentration of isoprenaline was also increased significantly by PTX in TGbeta(2) mice (9.40+/-1.22%, n=8) and was no longer reduced compared to LM mice (8.93+/-1.50%, n=11). Both beta(1)- and beta(2)AR subtypes were affected both by the original desensitization and by the resensitization with PTX. PTX treatment has therefore restored the original phenotype, with high basal contractility and little further effect of isoprenaline. We suggest that both beta-AR desensitization and lack of increased basal contraction in ventricular myocytes from our colony of TGbeta(2) mice were due to increased activity of PTX-sensitive G-proteins.

The objective of this study was to determine how maturation and aging affects beta (beta)-adrenergic receptor (AR) control of arterial vasorelaxation. Left ventricular (LV) hemodynamics and arterial vasorelaxation in thoracic artery segments were studied in Brown Norway, Fisher 344 cross rats at 6 weeks, 6 months, and 23 months of age. We defined changes in maturation as occurring between 6 weeks and 6 months of age and changes in aging as occurring between 6 months and 23 months of age. With maturation, isoproterenol resulted in a downward shift in heart rate and an upward shift in both LV dP/dt and peripheral vascular resistance responses. Similar changes were noted with aging except for the downward shift in LV dP/dt isoproterenol response. There was a dose-dependent increase in arterial vasorelaxation in response to isoproterenol in all age groups, but the 6-week-old animals had a 5-fold (P<0.01) increase in vasorelaxation compared to other age groups. The isoproterenol-induced arterial vasorelaxation response was not altered by removal of the endothelium. The vasodilatory responses to nitroglycerin, acetylcholine, and adenosine were diminished (P<0.05) with aging. The vasorelaxation responses to forskolin and IBMX were unchanged with maturation and diminished with aging. Incubation of arterial rings in cholera toxin resulted in a reduction in relaxation only in arteries from 6-week-old rats. Maturation resulted in no change in beta -AR density [20.2+/-0.7 v. 18.5+/-0.5 fmol/mg protein, P=n.s., 6 weeks (n=2, 18 aortas were combined v 6-month-old rats)]. With maturation, there was no change in G alpha(i)level. However, beta ARK1 levels were increased (55. 4+/-2.1 v. 40.8+/-0.4, arbitrary densitometry units) and G alpha(s)levels were decreased (29.5+/-0.8 v. 49.9+/-1.9, arbitrary densitometry units). Aging resulted in no change in beta -AR density (15.3+/-1.7 v. 18.5+/-0.5 fmol/mg membrane protein), but decreases in basal, isoproterenol-, naF-, and forskolin-stimulated AC activities. Compared to 6 week data, 23-month-old rats exhibited no change in either G alpha(i)or beta ARK1, however, G alpha(s) was decreased. In summary, beta -AR-stimulated arterial vasorelaxation is depressed during maturation and aging. Since there is no change in beta -AR density but a decrease in G alpha(s)and in basal/stimulated AC activities, the defect in beta -AR signaling during maturation and aging is probably a post receptor defect, i.e. possibly in the receptor-G protein coupling., (Copyright 2000 Academic Press.)

Objectives: Ex vivo perfusion of the cardiac allograft during organ procurement is an ideal environment for adenoviral vectors with transgenes that target improving graft contractility. One such target is the beta-adrenergic receptor-signaling system, in which alterations in transgenic mice have elucidated novel means to improve the function of the heart in vivo. The purpose of the current study was to determine the functional consequences of beta-adrenergic receptor manipulation in a rabbit model of cardiac allograft transplantation., Methods: New Zealand White rabbits weighing 3 kg served as recipients to 1-kg outbred donors. Donor hearts were arrested and harvested, and 1 of 3 adenoviral constructs was administered into the aortic root perfusing the graft. Transgenes delivered encoded either the human beta(2)-adrenergic receptor, a peptide inhibitor of beta-adrenergic receptor densensitization, or the marker transgene beta-galactosidase., Results: Five days after cervical heterotopic transplantation, left ventricular performance was measured on a Langendorff apparatus. A moderate pattern of rejection was seen in all grafts. Biventricular myocyte expression of beta-galactosidase was observed, and beta(2)-adrenergic receptor density was elevated 10-fold in grafts that received adeno-beta(2)-adrenergic receptor. Left ventricular systolic and diastolic performance was significantly increased in grafts transfected with either adeno-beta(2)-adrenergic receptor or adeno-beta-adrenergic receptor densensitization compared with control grafts that received adeno-beta-galactosidase., Conclusions: Ex vivo adenovirus-mediated gene transfer is feasible in a rabbit allograft model and, more important, genetic manipulation of beta-adrenergic receptor signaling either by increasing beta(2)-adrenergic receptor density or blocking endogenous receptor desensitization improves graft function acutely in this allograft model.

Objective: Right ventricular dysfunction is a poorly understood but persistent clinical problem. This study was undertaken to evaluate ventricular performance and beta-adrenergic receptor signaling in a tricuspid regurgitation model of right ventricular overload., Methods: Seventeen dogs were chronically instrumented with epicardial dimension transducers. By means of the shell-subtraction model, right ventricular pressure-volume relationships were evaluated in normal and right ventricular overload states. Right ventricular chamber performance was quantified by the stroke work at an end-diastolic volume relationship., Results: Right ventricular volume overload caused a 28% +/- 11% and 31% +/- 9% decline in chamber performance acutely and at 1 week, respectively, whereas end-diastolic volume increased from 45 +/- 21 to 60 +/- 30 mL (P =. 019). beta-Adrenergic receptor signaling in myocardial samples was assessed, examining adenylyl cyclase and G-protein-coupled receptor kinase activity. Stimulated adenylyl cyclase activity significantly decreased, and G-protein-coupled receptor kinase activity significantly increased in both left and right ventricular samples caused by increased levels of beta-adrenergic receptor kinase 1. No change in beta-adrenergic receptor density was seen at 1 week., Conclusions: Early right ventricular overload is associated with impaired right ventricular chamber contractility, dilation, and, importantly, a biventricular alteration of beta-adrenergic receptor signaling.

Cardiac-specific overexpression of the human beta(2)-adrenergic receptor (AR) in transgenic mice (TG4) enhances basal cardiac function due to ligand-independent spontaneous beta(2)-AR activation. However, agonist-mediated stimulation of either beta(1)-AR or beta(2)-AR fails to further enhance contractility in TG4 ventricular myocytes. Although the lack of beta(2)-AR response has been ascribed to an efficient coupling of the receptor to pertussis toxin-sensitive G(i) proteins in addition to G(s), the contractile response to beta(1)-AR stimulation by norepinephrine and an alpha(1)-adrenergic antagonist prazosin is not restored by pertussis toxin treatment despite a G(i) protein elevation of 1.7-fold in TG4 hearts. Since beta-adrenergic receptor kinase, betaARK1, activity remains unaltered, the unresponsiveness of beta(1)-AR is not caused by betaARK1-mediated receptor desensitization. In contrast, pre-incubation of cells with anti-adrenergic reagents such as muscarinic receptor agonist, carbachol (10(-5)m), or a beta(2)-AR inverse agonist, ICI 118,551 (5 x 10(-7)m), to abolish spontaneous beta(2)-AR signaling, both reduce the base-line cAMP and contractility and, surprisingly, restore the beta(1)-AR contractile response. The "rescued" contractile response is completely reversed by a beta(1)-AR antagonist, CGP 20712A. Furthermore, these results from the transgenic animals are corroborated by in vitro acute gene manipulation in cultured wild type adult mouse ventricular myocytes. Adenovirus-directed overexpression of the human beta(2)-AR results in elevated base-line cAMP and contraction associated with a marked attenuation of beta(1)-AR response; carbachol pretreatment fully revives the diminished beta(1)-AR contractile response. Thus, we conclude that constitutive beta(2)-AR activation induces a heterologous desensitization of beta(1)-ARs independent of betaARK1 and G(i) proteins; suppression of the constitutive beta(2)-AR signaling by either a beta(2)-AR inverse agonist or stimulation of the muscarinic receptor rescues the beta(1)-ARs from desensitization, permitting agonist-induced contractile response.

When the heart fails, there is often a constellation of biochemical alterations of the beta-adrenergic receptor (betaAR) signaling system, leading to the loss of cardiac inotropic reserve. betaAR down-regulation and functional uncoupling are mediated through enhanced activity of the betaAR kinase (betaARK1), the expression of which is increased in ischemic and failing myocardium. These changes are widely viewed as representing an adaptive mechanism, which protects the heart against chronic activation. In this study, we demonstrate, using in vivo intracoronary adenoviral-mediated gene delivery of a peptide inhibitor of betaARK1 (betaARKct), that the desensitization and down-regulation of betaARs seen in the failing heart may actually be maladaptive. In a rabbit model of heart failure induced by myocardial infarction, which recapitulates the biochemical betaAR abnormalities seen in human heart failure, delivery of the betaARKct transgene at the time of myocardial infarction prevents the rise in betaARK1 activity and expression and thereby maintains betaAR density and signaling at normal levels. Rather than leading to deleterious effects, cardiac function is improved, and the development of heart failure is delayed. These results appear to challenge the notion that dampening of betaAR signaling in the failing heart is protective, and they may lead to novel therapeutic strategies to treat heart disease via inhibition of betaARK1 and preservation of myocardial betaAR function.

Activation of phosphoinositide 3-kinases is coupled to both phosphotyrosine/growth factor and G protein-coupled receptors. We explored the role of phosphoinositide 3-kinase activation in myocardium during in vivo pressure overload hypertrophy in mice. Cytosolic extracts from wild type hypertrophied hearts showed a selective increase in the phosphoinositide 3-kinase gamma isoform. To address the role of G protein-coupled receptor-mediated activation of phosphoinositide 3-kinase, we used transgenic mice with cardiac-specific overexpression of a Gbetagamma sequestering peptide. Extracts from hypertrophied transgenic hearts showed complete loss of phosphoinositide 3-kinase activation, indicating a Gbetagamma-dependent process. To determine the class of G proteins that contribute Gbetagamma dimers for in vivo phosphoinositide 3-kinase activation, two strategies were used: 1) transgenic mice with cardiac-specific overexpression of a G(q) inhibitor peptide and 2) pertussis toxin treatment prior to pressure overload in wild type mice. Pressure overloaded G(q) inhibitor transgenic mice showed a complete absence of phosphoinositide 3-kinase activation, whereas pretreatment with pertussis toxin showed robust phosphoinositide 3-kinase activation. Taken together, these data demonstrate that activation of the phosphoinositide 3-kinase during in vivo pressure overload hypertrophy is Gbetagamma-dependent and the Gbetagamma dimers arise from stimulation of G(q)-coupled receptors.

Background: Genetic modulation of ventricular function may offer a novel therapeutic strategy for patients with congestive heart failure. Myocardial overexpression of beta(2)-adrenergic receptors (beta(2)ARs) has been shown to enhance contractility in transgenic mice and reverse signaling abnormalities found in failing cardiomyocytes in culture. In this study, we sought to determine the feasibility and in vivo consequences of delivering an adenovirus containing the human beta(2)AR cDNA to ventricular myocardium via catheter-mediated subselective intracoronary delivery., Methods and Results: Rabbits underwent percutaneous subselective catheterization of either the left or right coronary artery and infusion of adenoviral vectors containing either a marker transgene (Adeno-betaGal) or the beta(2)AR (Adeno-beta(2)AR). Ventricular function was assessed before catheterization and 3 to 6 days after gene delivery. Both left circumflex- and right coronary artery-mediated delivery of Adeno-beta(2)AR resulted in approximately 10-fold overexpression in a chamber-specific manner. Delivery of Adeno-betaGal did not alter in vivo left ventricular (LV) systolic function, whereas overexpression of beta(2)ARs in the LV improved global LV contractility, as measured by dP/dt(max), at baseline and in response to isoproterenol at both 3 and 6 days after gene delivery., Conclusions: Percutaneous adenovirus-mediated intracoronary delivery of a potentially therapeutic transgene is feasible, and acute global LV function can be enhanced by LV-specific overexpression of the beta(2)AR. Thus, genetic modulation to enhance the function of the heart may represent a novel therapeutic strategy for congestive heart failure and can be viewed as molecular ventricular assistance.

G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors, including alpha(1B)-adrenergic receptors (ARs), resulting in desensitization. In vivo analysis of GRK substrate selectivity has been limited. Therefore, we generated hybrid transgenic mice with myocardium-targeted overexpression of 1 of 3 GRKs expressed in the heart (GRK2 [commonly known as the beta-AR kinase 1], GRK3, or GRK5) with concomitant cardiac expression of a constitutively activated mutant (CAM) or wild-type alpha(1B)AR. Transgenic mice with cardiac CAMalpha(1B)AR overexpression had enhanced myocardial alpha(1)AR signaling and elevated heart-to-body weight ratios with ventricular atrial natriuretic factor expression denoting myocardial hypertrophy. Transgenic mouse hearts overexpressing only GRK2, GRK3, or GRK5 had no hypertrophy. In hybrid transgenic mice, enhanced in vivo signaling through CAMalpha(1B)ARs, as measured by myocardial diacylglycerol content, was attenuated by concomitant overexpression of GRK3 but not GRK2 or GRK5. CAMalpha(1B)AR-induced hypertrophy and ventricular atrial natriuretic factor expression were significantly attenuated with either concurrent GRK3 or GRK5 overexpression. Similar GRK selectivity was seen in hybrid transgenic mice with wild-type alpha(1B)AR overexpression concurrently with a GRK. GRK2 overexpression was without effect on any in vivo CAM or wild-type alpha(1B)AR cardiac phenotype, which is in contrast to previously reported in vitro findings. Furthermore, endogenous myocardial alpha(1)AR mitogen-activated protein kinase signaling in single-GRK transgenic mice also exhibited selectivity, as GRK3 and GRK5 desensitized in vivo alpha(1)AR mitogen-activated protein kinase responses that were unaffected by GRK2 overexpression. Thus, these results demonstrate that GRKs differentially interact with alpha(1B)ARs in vivo such that GRK3 desensitizes all alpha(1B)AR signaling, whereas GRK5 has partial effects and, most interestingly, GRK2 has no effect on in vivo alpha(1B)AR signaling in the heart.

From the ability to successfully manipulate the mouse genome has come important transgenic and gene-targeted knockout models that impact many areas of biomedical research. Genetically engineered mouse models geared toward the study of cardiovascular regulation have recently been described and provide powerful tools to study normal and compromised cardiac physiology. The genetic manipulation of the adrenergic receptor (AR) signaling system in the heart, including its regulation by desensitizing kinases, has shed light on the role of this signaling pathway in the regulation of cardiac contractility. One major finding, supported by several mouse models, is that in vivo contractility can be enhanced via alteration of myocardial AR signaling. Thus genetic manipulation of this critical receptor system in the heart represents a novel therapeutic approach for improving function of the failing heart.

Cardiac beta(2)-adrenergic receptor (beta(2)AR) overexpression is a potential contractile therapy for heart failure. Cardiac contractility was elevated in mice overexpressing beta(2)ARs (TG4s) with no adverse effects under normal conditions. To assess the consequences of beta(2)AR overexpression during ischemia, perfused hearts from TG4 and wild-type mice were subjected to 20-minute ischemia and 40-minute reperfusion. During ischemia, ATP and pH fell lower in TG4 hearts than wild type. Ischemic injury was greater in TG4 hearts, as indicated by lower postischemic recoveries of contractile function, ATP, and phosphocreatine. Because beta(2)ARs, unlike beta(1)ARs, couple to G(i) as well as G(s), we pretreated mice with the G(i) inhibitor pertussis toxin (PTX). PTX treatment increased basal contractility in TG4 hearts and abolished the contractile resistance to isoproterenol. During ischemia, ATP fell lower in TG4+PTX than in TG4 hearts. Recoveries of contractile function and ATP were lower in TG4+PTX than in TG4 hearts. We also studied mice that overexpressed either betaARK1 (TGbetaARK1) or a betaARK1 inhibitor (TGbetaARKct). Recoveries of function, ATP, and phosphocreatine were higher in TGbetaARK1 hearts than in wild-type hearts. Despite basal contractility being elevated in TGbetaARKct hearts to the same level as that of TG4s, ischemic injury was not increased. In summary, beta(2)AR overexpression increased ischemic injury, whereas betaARK1 overexpression was protective. Ischemic injury in the beta(2)AR overexpressors was exacerbated by PTX treatment, implying that it was G(s) not G(i) activity that enhanced injury. Unlike beta(2)AR overexpression, basal contractility was increased by betaARK1 inhibitor expression without increasing ischemic injury, thus implicating a safer potential therapy for heart failure.

The beta-adrenergic signaling cascade is an important regulator of myocardial function. Significant alterations of this pathway are associated with several cardiovascular diseases, including congestive heart failure (CHF). Included in these alterations is increased activity and expression of G protein-coupled receptor kinases (GRKs), such as the beta-adrenergic receptor kinase (beta ARK1), which phosphorylate and desensitize beta-adrenergic receptors (beta ARs). A body of evidence is accumulating that suggests that GRKs, in particular beta ARK1, are critical determinants of cardiac function under normal conditions and in disease states. Transgenic mice with myocardial-targeted alterations of GRK activity have shown profound changes in the in vivo functional performance of the heart. Included in these studies is the compelling finding that inhibition of beta ARK1 activity or expression significantly enhances cardiac function and potentiates beta AR signaling in failing cardiomyocytes. This article summarizes the advances made in the study of beta ARK1 in the heart and addresses its potential as a novel therapeutic target for CHF.

Congestive heart failure (CHF), despite the improved prevention and treatment modalities adopted for cardiovascular disease over the past two decades, remains a significant therapeutic challenge. Efficacious therapies are few, and death rates from CHF continue to rise. Recent advances in our understanding of the molecular basis of CHF have given rise to experimental animal models demonstrating related genetic phenotypes, which further elucidate cellular mechanisms involved in the pathogenesis of the failing heart. Studies involving transgenic mice have elucidated novel potential gene therapy interventions aimed at the genetic modification of beta-adrenergic signaling in the heart. This review will briefly discuss beta-adrenergic signaling in CHF, while focusing on potential gene therapy strategies to improve the performance of the failing heart.

Proliferation of vascular smooth muscle (VSM) severely affects the outcome of coronary artery bypass and angioplasty procedures, causing the failure of venous grafts or restenosis of the reopened vessel. Investigation into the mechanisms underlying the process of VSM cellular proliferation has provided evidence that intracellular signaling mechanisms triggered by extracellular hormonal factors acting through G protein-coupled receptors, can mediate and sustain this pathological process. Inhibition of common pathways of G protein-coupled receptor signaling has recently proven effective in preventing VSM cellular activation and proliferation. In particular, inhibition of the G beta gamma-mediated mitogen-activated protein (MAP) kinase signaling pathway results in the inhibition of VSM proliferation in vitro. Moreover, use of adenoviral vectors to deliver a peptide inhibitor of G beta gamma signaling in vivo has resulted in inhibition of intimal hyperplasia in experimental models of vein-graft failure and restenosis.

Background: The clinical syndrome of heart failure (HF) is characterized by an impaired cardiac beta-adrenergic receptor (betaAR) system, which is critical in the regulation of myocardial function. Expression of the betaAR kinase (betaARK1), which phosphorylates and uncouples betaARs, is elevated in human HF; this likely contributes to the abnormal betaAR responsiveness that occurs with beta-agonist administration. We previously showed that transgenic mice with increased myocardial betaARK1 expression had impaired cardiac function in vivo and that inhibiting endogenous betaARK1 activity in the heart led to enhanced myocardial function., Methods and Results: We created hybrid transgenic mice with cardiac-specific concomitant overexpression of both betaARK1 and an inhibitor of betaARK1 activity to study the feasibility and functional consequences of the inhibition of elevated betaARK1 activity similar to that present in human HF. Transgenic mice with myocardial overexpression of betaARK1 (3 to 5-fold) have a blunted in vivo contractile response to isoproterenol when compared with non-transgenic control mice. In the hybrid transgenic mice, although myocardial betaARK1 levels remained elevated due to transgene expression, in vitro betaARK1 activity returned to control levels and the percentage of betaARs in the high-affinity state increased to normal wild-type levels. Furthermore, the in vivo left ventricular contractile response to betaAR stimulation was restored to normal in the hybrid double-transgenic mice., Conclusions: Novel hybrid transgenic mice can be created with concomitant cardiac-specific overexpression of 2 independent transgenes with opposing actions. Elevated myocardial betaARK1 in transgenic mouse hearts (to levels seen in human HF) can be inhibited in vivo by a peptide that can prevent agonist-stimulated desensitization of cardiac betaARs. This may represent a novel strategy to improve myocardial function in the setting of compromised heart function.

Calsequestrin is a high capacity Ca(2+)-binding protein in the junctional sarcoplasmic reticulum that forms a quaternary complex with junctin, triadin, and the ryanodine receptor. Transgenic mice with cardiac-targeted calsequestrin overexpression show marked suppression of Ca(2+)-induced Ca(2+) release, myocyte hypertrophy, and premature death by 16 weeks of age (Jones, L. R., Suzuki, Y. J., Wang, W., Kobayashi, Y. M., Ramesh, V., Franzini-Armstrong, C., Cleemann, L., and Morad, M. (1998) J. Clin. Invest. 101, 1385-1393). To investigate whether alterations in intracellular Ca(2+) trigger changes in the beta-adrenergic receptor pathway, we studied calsequestrin overexpressing transgenic mice at 7 and 14 weeks of age. As assessed by echocardiography, calsequestrin mice at 7 weeks showed mild left ventricular enlargement, mild decreased fractional shortening with increased wall thickness. By 14 weeks, the phenotype progressed to marked left ventricular enlargement and severely depressed systolic function. Cardiac catheterization in calsequestrin mice revealed markedly impaired beta-adrenergic receptor responsiveness in both 7- and 14- week mice. Biochemical analysis in 7- and 14-week mice showed a significant decrease in total beta-adrenergic receptor density, adenylyl cyclase activity, and the percent high affinity agonist binding, which was associated with increased beta-adrenergic receptor kinase 1 levels. Taken together, these data indicate that alterations in beta-adrenergic receptor signaling precede the development of overt heart failure in this mouse model of progressive cardiomyopathy.

Background: Intimal hyperplasia remains the leading cause of vein graft failure. Various external stenting devices have been shown to reduce the development of intimal hyperplasia in vein grafts. Mitogenic and mechanotransduction signals are known to be mediated by G protein-coupled receptors. Therefore in this study we examined the alterations in G protein expression and receptor coupling in vein grafts stented with external tube support., Methods: Thirty New Zealand White male rabbits had a right carotid interposition bypass graft with use of the ipsilateral jugular vein. Fifteen animals received external support and 15 were controls. In a subset the animals either had removal of the external support or a sham-control neck exploration at 14 days after the initial implantation (n = 5 per group)., Results: External support reduced G alpha i3 proteins by 30% in vein grafts without changes in G alpha s by Western blot. Vein grafts with external support were significantly less sensitive to pertussis toxin inactivation than controls were in response to both norepinephrine and serotonin. A 24% decrease in intimal thickness was maintained after withdrawal of the initial external support., Conclusions: The placement of an external support is associated with alternations in G protein expression and receptor coupling function in vein grafts. The results of this study suggest that the development of vein graft intimal hyperplasia may involve G protein-mediated events.