Therapeutic safety of high myocardial expression levels of the molecular inotrope S100A1 in a preclinical heart failure model.

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.

Myocardial ß(2) -adrenoceptor gene delivery promotes coordinated cardiac adaptive remodelling and angiogenesis in heart failure.

BACKGROUND AND PURPOSE: We investigated whether ß(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 ß(2) -adrenoceptor overexpression in a rat model of post-myocardial infarction (MI) heart failure (HF). Cardiac adenoviral-mediated ß(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 ß(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 ß(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-ß(2) -adrenoceptor HF rats showed improved LV remodeling and cardiac function. Importantly, ß(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 ß(2) -adrenoceptor gene transfer induced the activation of the VEGF/PKB/eNOS pro-angiogenic pathway. In ß(2) -adrenoceptor-/- mice, we found a ~25% reduction in cardiac capillary density compared with ß(2) -adrenoceptor+/+ mice. The lack of ß(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: ß(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.

Targeting GRK2 by gene therapy for heart failure: benefits above ß-blockade.

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 ß-adrenergic receptor antagonism.

Gene therapy targets in heart failure: the path to translation.

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 ß-adrenergic receptor (ß-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.

Myocardial gene delivery using molecular cardiac surgery with recombinant adeno-associated virus vectors in vivo.

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.

Developing strategies to link basic cardiovascular sciences with clinical drug development: another opportunity for translational sciences.

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 gene transfer in myocardium.

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.

S100A1 increases the gain of excitation-contraction coupling in isolated rabbit ventricular cardiomyocytes.

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.

The adrenergic pathway and heart failure.

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.

A porcine model of intimal-medial hyperplasia in polytetrafluoroethylene arteriovenous grafts.

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.

Adenoviral gene transfer to the heart during cardiopulmonary bypass: effect of myocardial protection technique on transgene expression.

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.

Gene-mediated inhibition of the b-adrenergic receptor kinase: a new therapeutic strategy for heart failure.

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 regulator of myocardial contractility.

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.

Right ventricular gene therapy with a beta-adrenergic receptor kinase inhibitor improves survival after pulmonary artery banding.

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.

Genetic manipulation of beta-adrenergic signalling in heart failure.

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.

Ventricular dysfunction after cardioplegic arrest is improved after myocardial gene transfer of a beta-adrenergic receptor kinase inhibitor.

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.

Transgenic studies of cardiac adrenergic receptor regulation.

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.

Cardiac betaARK1 upregulation induced by chronic salt deprivation in rats.

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.

Posttraumatic stress disorder arising after road traffic collisions: patterns of response to cognitive-behavior therapy.

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.

Regulation of myocardial betaARK1 expression in catecholamine-induced cardiac hypertrophy in transgenic mice overexpressing alpha1B-adrenergic receptors.

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.