This study examines transgene expression and biodistribution of adeno-associated virus (AAV) pseudotyped 1-9 after tail vein (TV) injection in male mice. Using a cytomegalovirus (CMV)-luciferase transgene, the time-course of expression in each animal was tracked throughout the experiment. The animals were imaged at 7, 14, 29, 56, and 100 days after the TV injection. The total number of photons emitted from each animal was recorded, allowing examination of expression level and kinetics for each pseudotyped virus. The bioluminescence imaging revealed three expression levels (i) low-expression group, AAV2, 3, 4, and 5; (ii) moderate-expression group, AAV1, 6, and 8; and (iii) high-expression group, AAV7 and 9. In addition, imaging revealed two classes of kinetics (i) rapid-onset, for AAV1, 6, 7, 8, and 9; and (ii) slow-onset, for AAV2, 3, 4, and 5. We next evaluated protein expression and viral genome copy numbers in dissected tissues. AAV9 had the best viral genome distribution and highest protein levels. The AAV7 protein and genome copy numbers were comparable to those of AAV9 in the liver. Most surprisingly, AAV4 showed the greatest number of genome copies in lung and kidney, and a high copy number in the heart. AAV6 expression was observed in the heart, liver, and skeletal muscle, and the genome distribution corroborated these observations.

Background-The upregulation of G protein-coupled receptor kinase 2 in failing myocardium appears to contribute to dysfunctional ␤-adrenergic receptor (␤AR) signaling and cardiac function. The peptide ␤ARKct, which can inhibit the activation of G protein-coupled receptor kinase 2 and improve ␤AR signaling, has been shown in transgenic models and short-term gene transfer experiments to rescue heart failure (HF). This study was designed to evaluate long-term ␤ARKct expression in HF with the use of stable myocardial gene delivery with adeno-associated virus serotype 6 (AAV6). Methods and Results-In HF rats, we delivered ␤ARKct or green fluorescent protein as a control via AAV6-mediated direct intramyocardial injection. We also treated groups with concurrent administration of the ␤-blocker metoprolol. We found robust and long-term transgene expression in the left ventricle at least 12 weeks after delivery. ␤ARKct significantly improved cardiac contractility and reversed left ventricular remodeling, which was accompanied by a normalization of the neurohormonal (catecholamines and aldosterone) status of the chronic HF animals, including normalization of cardiac ␤AR signaling. Addition of metoprolol neither enhanced nor decreased ␤ARKct-mediated beneficial effects, although metoprolol alone, despite not improving contractility, prevented further deterioration of the left ventricle. Conclusions-Long-term cardiac AAV6-␤ARKct gene therapy in HF results in sustained improvement of global cardiac function and reversal of remodeling at least in part as a result of a normalization of the neurohormonal signaling axis. In addition, ␤ARKct alone improves outcomes more than a ␤-blocker alone, whereas both treatments are compatible. These findings show that ␤ARKct gene therapy can be of long-term therapeutic value in HF. (Circulation. 2009;119:89-98.)

Our data indicate that exercise favourably affects angiogenesis and improves LV remodelling and contractility reserve in a rat model of severe chronic HF.

Aldosterone produces a multitude of effects in vivo, including promotion of postmyocardial infarction adverse cardiac remodeling and heart failure progression. It is produced and secreted by the adrenocortical zona glomerulosa (AZG) cells after angiotensin II (AngII) activation of AngII type 1 receptors (AT1Rs). Until now, the general consensus for AngII signaling to aldosterone production has been that it proceeds via activation of Gq/11-proteins, to which the AT1R normally couples. Here, we describe a novel signaling pathway underlying this AT1R-dependent aldosterone production mediated by ␤-arrestin-1 (␤arr1), a universal heptahelical receptor adapter/scaffolding protein. This pathway results in sustained ERK activation and subsequent up-regulation of steroidogenic acute regulatory protein, a steroid transport protein regulating aldosterone biosynthesis in AZG cells. Also, this ␤arr1-mediated pathway appears capable of promoting aldosterone turnover independently of G protein activation, because treatment of AZG cells with SII, an AngII analog that induces ␤arr, but not G protein coupling to the AT1R, recapitulates the effects of AngII on aldosterone production and secretion. In vivo, increased adrenal ␤arr1 activity, by means of adrenal-targeted adenoviral-mediated gene delivery of a ␤arr1 transgene, resulted in a marked elevation of circulating aldosterone levels in otherwise normal animals, suggesting that this adrenocortical ␤arr1-mediated signaling pathway is operative, and promotes aldosterone production and secretion in vivo, as well. Thus, inhibition of adrenal ␤arr1 activity on AT1Rs might be of therapeutic value in pathological conditions characterized and aggravated by hyperaldosteronism.adrenocortical zona glomerulosa cell ͉ G protein-coupled receptor ͉ angiotensin II receptor type I ͉ adrenal steroid hormones ͉ biased agonism

Cardiac gene transfer is an attractive tool for developing novel heart disease treatments. Adeno-associated viral (AAV) vectors are widely used to mediate transgene expression in animal models and are being evaluated for human gene therapy. However, it is not clear which serotype displays the best cardiac tropism. Therefore, we curried out this study to directly compare AAV serotypes 1–9 heart transduction efficiency after indirect intracoronary injection. AAV-CMV-luciferase serotypes 1–9 were injected in the left ventricular cavity of adult mice, after cross-clamping the ascending aorta and pulmonary artery. An imaging system was used to visualize luciferase expression at 3-7-21-70-140 days post-injection. Echocardiography was performed to evaluate cardiac function on day 140. At the end of the study luciferase enzyme activity and genome copies of the different AAV serotypes were assessed in several tissues and potential AAV immunogenicity was evaluated on heart sections by staining for macrophage and lymphocyte antigens. Among AAV serotypes 1–9, AAV6 showed the best capability of achieving high transduction levels in the myocardium in a tissue-specific manner, whereas the other serotypes owned less cardiac transduction and more extra-cardiac expression, especially in the liver. Importantly, none of the serotypes tested with this marker gene affected cardiac function nor was associated with inflammation.

Background The Sphingosine-1-phosphate receptor 1 (S1PR1) and β1-adrenergic receptor (β1AR) are G protein-coupled receptors (GPCRs) expressed in the heart. These two GPCRs have opposing actions on adenylyl cyclase due to differential G protein-coupling. Importantly, both of these receptors can be regulated by the actions of GPCR kinase-2 (GRK2), which triggers desensitization and down-regulation processes. Although, classical signaling paradigms suggest that simultaneous activation of β1ARs and S1PR1s in a myocyte would simply be opposing action on cAMP production, in this report we have uncovered a direct interaction between these two receptors with a regulatory involvement of GRK2. Methods and Results In HEK293 cells overexpressing both β1AR and S1PR1, we demonstrate that β1AR down-regulation can occur after sphingosine 1-phosphate (S1PR1 agonist) stimulation while S1PR1 down-regulation can be triggered by isoproterenol (βAR agonist) treatment. This cross-talk between these two distinct GPCRs appears to have physiological significance since they interact and show reciprocal regulation in mouse hearts undergoing chronic βAR stimulation and also in a rat model of post-ischemic heart failure (HF). Conclusions We demonstrate that restoring cardiac plasma membrane levels of S1PR1 produce beneficial effects counterbalancing deleterious β1AR overstimulation in HF.

Objectives We investigated whether adrenal beta-arrestin 1 (βarr1)-mediated aldosterone production plays any role in post-myocardial infarction (MI) heart failure (HF) progression. Background Heart failure represents 1 of the most significant health problems worldwide, and new and innovative treatments are needed. Aldosterone contributes significantly to HF progression after MI by accelerating adverse cardiac remodeling and ventricular dysfunction. It is produced by the adrenal cortex after angiotensin II activation of angiotensin II type 1 receptors (AT1Rs), G protein-coupled receptors that also signal independently of G proteins. The G protein-independent signaling is mediated by βarr1 and βarr2. We recently reported that adrenal βarr1 promotes AT1R-dependent aldosterone production leading to elevated circulating aldosterone levels in vivo. Methods Adrenal-targeted, adenoviral-mediated gene delivery in vivo in 2-week post-MI rats, a time point around which circulating aldosterone significantly increases to accelerate HF progression, was performed to either increase the expression of adrenal βarr1 or inhibit its function via expression of a βarr1 C-terminal-derived peptide fragment. Results We found that adrenal βarr1 overexpression promotes aldosterone elevation after MI, resulting in accelerated cardiac adverse remodeling and deterioration of ventricular function. Importantly, these detrimental effects of aldosterone are prevented when adrenal βarr1 is inhibited in vivo, which markedly decreases circulating aldosterone after MI. Finally, the prototypic AT1R antagonist losartan seems unable to lower this adrenal βarr1-driven aldosterone elevation. Conclusions Adrenal βarr1 inhibition, either directly or with AT1R “biased” antagonists that prevent receptor-βarr1 coupling, might be of therapeutic value for curbing HF-exacerbating hyperaldosteronism.

We recently reported that the upregulation of adrenal G protein-coupled receptor kinase-2 (GRK2) causes enhanced catecholamine (CA) secretion by desensitizing sympatho-inhibitory alpha (2)-adrenergic receptors (alpha (2)ARs) of chromaffin cells, and thereby aggravating heart failure (HF). In this study, we sought to develop an efficient and reproducible in vivo adrenal gene transfer method to determine whether manipulation of adrenal GRK2 levels/activity regulates physiological CA secretion in rats. We specifically investigated two different in vivo gene delivery methods: direct injection into the suprarenal glands, and retrograde delivery through the suprarenal veins. We delivered adenoviral (Ad) vectors containing either GRK2 or an inhibitor of GRK2 activity, the beta ARKct. We found both delivery approaches equally effective at supporting robust (>80% of the whole organ) and adrenal-restricted transgene expression, in the cortical region as well as in the medullar region. Additionally, rats with AdGRK2-infected adrenals exhibit enhanced plasma CA levels when compared with control rats (AdGFP-injected adrenals), whereas plasma CA levels after Ad beta ARKct infection were significantly lower. Finally, in isolated chromaffin cells, alpha (2)ARs of AdGRK2-infected cells failed to inhibit CA secretion whereas Ad beta ARKct-infected cells showed normal alpha (2)AR responsiveness. These results not only indicate that in vivo adrenal gene transfer is an effective way of manipulating adrenal gland signalling, but also identify GRK2 as a critically important molecule involved in CA secretion.