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Background— The incidence of heart failure is ever-growing, and it is urgent to develop improved treatments. An attractive approach is gene therapy; however, the clinical barrier has yet to be broken because of several issues, including the lack of an ideal vector supporting safe and long-term myocardial transgene expression. Methods and Results— Here, we show that the use of a recombinant adeno-associated viral (rAAV6) vector containing a novel cardiac-selective enhancer/promoter element can direct stable cardiac expression of a therapeutic transgene, the calcium (Ca 2+ )-sensing S100A1, in a rat model of heart failure. The chronic heart failure–rescuing properties of myocardial S100A1 expression, the result of improved sarcoplasmic reticulum Ca 2+ handling, included improved contractile function and left ventricular remodeling. Adding to the clinical relevance, long-term S100A1 therapy had unique and additive beneficial effects over β-adrenergic receptor blockade, a current pharmacological heart failure treatment. Conclusions— These findings demonstrate that stable increased expression of S100A1 in the failing heart can be used for long-term reversal of LV dysfunction and remodeling. Thus, long-term, cardiac-targeted rAAV6-S100A1 gene therapy may be of potential clinical utility in human heart failure.

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 (AT 1 Rs). Until now, the general consensus for AngII signaling to aldosterone production has been that it proceeds via activation of G q/11 -proteins, to which the AT 1 R normally couples. Here, we describe a novel signaling pathway underlying this AT 1 R-dependent aldosterone production mediated by β-arrestin-1 (βarr1), 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 βarr1-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 βarr, but not G protein coupling to the AT 1 R, recapitulates the effects of AngII on aldosterone production and secretion. In vivo, increased adrenal βarr1 activity, by means of adrenal-targeted adenoviral-mediated gene delivery of a βarr1 transgene, resulted in a marked elevation of circulating aldosterone levels in otherwise normal animals, suggesting that this adrenocortical βarr1-mediated signaling pathway is operative, and promotes aldosterone production and secretion in vivo, as well. Thus, inhibition of adrenal βarr1 activity on AT 1 Rs might be of therapeutic value in pathological conditions characterized and aggravated by hyperaldosteronism.

Background— The upregulation of G protein–coupled receptor kinase 2 in failing myocardium appears to contribute to dysfunctional β-adrenergic receptor (βAR) signaling and cardiac function. The peptide βARKct, which can inhibit the activation of G protein–coupled receptor kinase 2 and improve βAR 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 βARKct 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 βARKct or green fluorescent protein as a control via AAV6-mediated direct intramyocardial injection. We also treated groups with concurrent administration of the β-blocker metoprolol. We found robust and long-term transgene expression in the left ventricle at least 12 weeks after delivery. βARKct 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 βAR signaling. Addition of metoprolol neither enhanced nor decreased βARKct-mediated beneficial effects, although metoprolol alone, despite not improving contractility, prevented further deterioration of the left ventricle. Conclusions— Long-term cardiac AAV6-βARKct 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, βARKct alone improves outcomes more than a β-blocker alone, whereas both treatments are compatible. These findings show that βARKct gene therapy can be of long-term therapeutic value in HF.

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.

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.