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Cardiovascular Research 2002 56(2):181-183; doi:10.1016/S0008-6363(02)00653-3
© 2002 by European Society of Cardiology
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Copyright © 2002, European Society of Cardiology

Conditional AC type VI expression in the heart: relevant insights into function of inducible target gene expression

Patrick Most, Andrew Remppis and Hugo A Katus*

Department of Medicine, Division of Cardiology, Angiology and Pneumology, University of Heidelberg, 69115 Heidelberg, Germany

* Corresponding author. Tel.: +49-622-156-8670; fax: +49-622-156-5516. hugo_katus{at}med.uni-heidelberg.de

Received 28 August 2002; accepted 28 August 2002

See article by Gao et al. [1] (pages 197–204) in this issue.

Manipulation of the mouse genome by transgenic approaches is a powerful tool to examine gene function as well as interaction of gene products in the intact animal [2–4]. In the past, gain-in-function models were the most frequently used transgenic animals to define the physiological relevance of gene products. In these animals, transgene expression is controlled by well characterized promoter elements that drive transgene expression by their cardio-specificity, while the onset of transgene expression depends on the temporal activation of the promotor employed [5–11]. However, the temporal regulation of transgene expression might prevent investigation of a number of important questions. In its worst case, the inappropriate onset of transgene expression can interfere with proper embryonal development resulting in early lethal phenotypes [12–14]. Less dramatically but more commonly, constitutive transcriptional activity of the promoter does not provide any information on developmental aspects of transgene expression [15–17] and prevents examination of the gene dosis and phenotype [18,19]. These limitations are overcome by conditional transgenic systems allowing control of the timing as well as the spatial pattern of gene expression (for reviews, see Refs. [20,21]).

To date a number of conditional gene expression systems have been introduced [22–26]. Among them the tetracycline-regulated binary system based on the tetracycline (tet) resistance operon of E. coli has emerged as the present system of choice for in vivo applications [27–29]. By the use of the {alpha}-myosin heavy chain ({alpha}-MHC) promotor this system has been successfully adapted to achieve cardiac-specific conditional gene expression in the heart [30,31]. As shown in Fig. 1, this system requires two lines of transgenic mice: one expressing a chimeric tet-regulateable transcriptional activator (tTA) in a cardiac-specific manner, and another carrying the target gene driven by a quiescent minimal cytomegaly virus promoter (CMVmin) which is flanked by multimerized copies of the tet operator sequences (tetO). By crossbreeding, double-transgenic progenies that harbour both regulatory elements are generated in which the transgene is expressed in the absence of tet but not in its presence (tet-off system). Taking advantage of the modular composition of most transcription factors, the transcriptional repression domain of the tet repressor has been replaced by a strong transactivation domain from the transcription factor VP16 of herpes simplex virus (HSV), while the DNA- and tet-binding domain have been retained. The resulting chimeric transcription factor (tTA), thus converted to a ligand-regulated transactivator, binds to the tetO sequences in the absence but not in the presence of tet. Subsequently, this protein strongly activates, rather than represses, transcription promoters including tetO sequences. Recently, the efficacy of this binary system has been successfully tested in the heart by Yu el al. [31].


Figure 1
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  		Fig. 1 Regulation of cardiac-directed target gene expression using the tet-off system. The cardiac-specific transactivator (tTA) uses cell-type-specific regulatory elements such as the {alpha}-MHC promoter to drive expression of the chimeric tTA. The cardiac-specific tTA consists of the DNA-binding domain (DNA BD) and the tetracycline binding domain (Tet BD) of the tet repressor combined with the transactivation domain from HSV of virion protein 16 (AD VP16). The target gene contains repetitive tet operator sequences (tetO) followed by a human minimal CMV promoter (CMVmin) and the gene of interest (Target). In the absence of tetracycline (Tet) or a derivative (doxycycline), the transactivator protein binds to the tetO sequences and activates target protein expression. Binding of Tet to the tTA protein inhibits target protein expression by reducing tTA affinity for the tetO sequences.

 
In the current issue of Cardiovascular Research, Gao et al. [1] report the generation of conditional transgenic mice with a cardiac-directed expression of murine adenylylcyclase type VI (ACVI). By taking advantage of the tight regulation provided by the tet-off system, the authors demonstrate ligand-controlled cardiac-specific expression of ACVI leading to a reversible change in contractile function. Effective down-regulation of ACVI gene expression was achieved by oral doxycyline administration with virtually no baseline transcription of ACVI from the tetO–CMVmin promoter while induction of target gene expression was attained by withdrawal of doxycyline from water supply. Unspecific effects of the tetracycline-derivative were carefully controlled. Four days after removal of tet suppression they first observed an increase of myocardial ACVI protein content leading to a 10-fold cardiac-restricted increase in ACVI protein levels within 10 days. Subsequent reconstitution of tet suppression effectively abolished target gene expression. In vivo half-life of induced ACVI protein ranged from 2 to 3 days. Although phenotypically silent in the unstressed organism, controlled overexpression of ACVI in these hearts lead to increased cardiac cAMP generation and augmented left ventricular contractility in response to acute β-adrenergic receptor (βAR) stimulation. Taking advantage of both traditional transgenic and adenoviral technology, Hammond and co-workers [32–34] previously demonstrated modulation of β-adrenergic-stimulated cAMP production and cardiac hemodynamics following cardiac-directed constitutive ACVI overexpression. They subsequently evaluated effects of cardiac-directed ACVI constitutive overexpression in a murine heart failure model secondary to overexpression of the signal transduction protein Gq. Importantly, crossbreeding of ACVI transgenic mice with the Gq heart failure model restored cAMP generating capacity, enhanced cardiac function as well as βAR responsiveness, abrogated myocardial hypertrophy and improved survival [35,36]. ACVI has therefore been claimed to be a novel target for heart failure therapy. But a ‘rescue’ of the abnormal phenotype by the crossbreeding experiments must be interpreted with caution since the expression of the target gene during growth and development may prevent the manifestation of index disease in the first place, while treatment of animals or humans with fully developed disease might not be beneficial.

Thus with regard to ACVI as a potential therapeutic agent for heart failure, the study demonstrates for the first time a ligand-controlled genetic manipulation of the heart that results in a reversible positive inotropic effect of a target gene in vivo. Now, by crossbreeding their conditional ACVI transgene with a myopathic transgenic they could evaluate the effects of ACVI when signs of heart failure are already present. It is noteworthy that ACVI overexpression mediated enhanced ‘recruitable’ cAMP responsiveness without sustained adrenergic activation which might account for the marked differences in phenotype when compared to the effects of prolonged overexpression of other members of the βAR-G protein–AC cascade, i.e. βAR or Gs{alpha} overexpression [37–39]. However, since the ACVI mediated ‘rescue’ of myocardial failure so far has been solely tested on a heart failure model with decreased AC activity, it is important to test whether ACVI gene transfer will be equally beneficial in heart failure models with more heterogenous defects displaying unchanged cAMP generation capacity.

In summary, the conditional transgene provided by Gao et al. [1] represents a very valuable contribution to the field of cardiovascular gene therapy. The exciting question of the therapeutic potential of ACVI as a novel target to treat human heart failure is still unresolved but the study moves one significant step closer to the sought answer.


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