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.)

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

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

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.