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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 19, 2003 as doi:10.1096/fj.02-0438fje. |
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* Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland, USA; and
Division of Molecular Cardiovascular Biology, Children’s Hospital Medical Center, Cincinnati, Ohio, USA
2Correspondence: Department of Physiology, University of Maryland School of Medicine, 655 W. Baltimore Street, Baltimore, MD 21201, USA. E-mail: ywang001{at}umaryland.edu
SPECIFIC AIMS
To investigate the functional role of stress-activated, c-Jun N-terminal kinase (JNK) in the development of heart failure, it is critical to establish a transgenic animal with tissue-specific and temporally regulated JNK activation in the heart. We tested an inducible cre-loxP-mediated DNA recombination approach to achieve cardiac-specific and temporally regulated gene-switch in an adult mouse heart. We investigated JNK activation in the adult transgenic heart upon induction of gene expression and the resulting cardiac phenotype.
PRINCIPAL FINDINGS
1. Generation of transgenic animal with floxed transgene construct
Transgenic mice were established using a FloxMKK7D construct that contained a 5.5-kb murine 
-myosin heavy chain (MHC) promoter followed by a green fluorescent protein (GFP) cDNA flanked (floxed) by two loxP sites. Further 3' to the GFP expression unit was a MKK7D cDNA, coding for a constitutively activated mutant of an upstream activator of JNK, followed by a SV40 poly A signal sequence. Four transgenic founders were identified, and offspring from one of them were used for this study. GFP expression was identified in all transgenic hearts (FloxMKK7D) by fluorescence microcopy of cardiac tissue as well as immunoblot analysis (data not shown). No cardiac abnormality was observed in the transgenic mice at 10 weeks of age.
2. Efficient gene-switch mediated by tamoxifen induced DNA recombination
To determine the recombination efficiency in the double-transgenic hearts, the expression levels of the GFP marker gene in MKK7D transgenic hearts were analyzed as an indicator of a recombination event in cardiac myocytes. As determined by immunoblot analysis, GFP protein was expressed at high levels in the hearts of single-transgenic FloxMKK7D mice or double-transgenic MKK7D mice without tamoxifen treatment (Fig. 1
A).However, GFP expression was markedly decreased in double-transgenic hearts treated with tamoxifen to near undetectable levels (Fig. 1A
). To corroborate the immunoblot result, direct observation of GFP fluorescence in the heart revealed strong and uniform GFP expressioin in atria and ventricle of the FloxMKK7D single-transgenic control heart. In contrast, double-transgenic hearts treated with tamoxifen displayed dramatically diminished GFP fluorescence except in a few remaining cells (Fig. 1B
). These data suggested that tamoxifen induced efficient Cre protein-mediated DNA recombination between the two loxP sites in the transgene construct.
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3. Induction of JNK activity in adult heart leads to cardiomyopathy
Deletion of the GFP expression unit in the FloxMKK7D construct initiates the expression of MKK7D. As a result, JNK activity was significantly induced in all double-transgenic hearts treated with tamoxifen as measured by antiphospho-JNK immunoblot analysis (Fig. 2
A). As expected from the function of MKK7D as an upstream kinase for JNK, induction of JNK activity was not a result of increased expression of total JNK protein (Fig. 2A
). Activation of JNK activity in the transgenic heart led to significant cardiac pathology compared with single-transgenic controls or untreated double-transgenic animals, characterized by robust induction of fetal gene expression (Fig. 2B
) and progressive enlargement of atrial size after tamoxifen induction. Ventricle size of the transgenic hearts remained unchanged after 8 weeks of JNK induction (left ventricle weight vs. body weight ratio in JNK-induced animals vs. controls, 2.68±0.55 vs. 2.84±0.25, P=0.6), suggesting that JNK activation in vivo is able to induce cardiac pathology in the absence of significant ventricular hypertrophy.
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CONCLUSIONS
In this report, we demonstrated that cre-loxP-mediated transgenesis was an efficient approach to achieve targeted and temporally regulated gene activation in the heart (Fig. 3
). Marker gene expression demonstrated that the DNA recombination induced by MCM protein was highly efficient and tamoxifen-dependent. Similar to an earlier report in ROSA mice, the gene-switch efficiency in this model was greater than 90% and was uniformly distributed in atria and ventricular myocardium. JNK activation as a result of DNA recombination led to significant cardiac pathology in an adult transgenic heart. The results represent the first in vivo evidence to link JNK activation directly with cardiac hypertrophy and pathology in adult hearts.
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Compared with tetracycline-dependent or other binary-inducible transgenic approaches, this strategy possesses several advantages, as well as limitations. First, cre-loxP-mediated gene manipulation is far more specific and robust in its induction. The basal level expression is eliminated by stop sequences, and little leakage in expression was detected in this system (Fig. 1)
. The fold of induction is mostly dependent on the promoter strength used to drive the transgene (in this case, the robust MHC promoter) and is less likely influenced by a surrounded genomic environment than a tetracycline-dependent promoter. Second, the floxed marker gene expression unit engineered in the transgenic construct not only serves as an effective stopper for target gene expression but also can be used to efficiently screen for expression levels and distribution among different transgenic lines. In light of the potential impact of GFP overexpression on cardiomyocytes in vivo, lacZ or luciferase cDNAs can be used as alternative marker genes to achieve the same objectives. This easy screening approach will help to eliminate transgenic lines that have no or low expression levels or abnormal distributions without the need of breeding with a second transgenic line, thus reducing the time and effort required to obtain desirable, double-transgenic mice for inducible target gene expression. Unlike inducible promoter systems, significant limitations in the potential application of a cre-loxP strategy are the lack of reversibility and graded induction for transgene expression. In addition, the cre-loxP-mediated DNA recombination event is a relatively slow process, taking days rather than hours to accomplish gene induction. Future development of a conditional transgenic system with a reliable and robust response along with refined regulation is still needed.
Although JNK, as a major stress-activated mitogen-activated protein kinase pathway, has been implicated in a variety of cardiac pathology, ranging from cardiac hypertrophy to ischemia/reperfusion injury, no direct in vivo evidence is available to support a link between JNK activation and specific features of heart failure. The investigation was complicated by the difficulty of establishing transgenic animals with targeted activation of JNK in the heart as a result of early lethality (Molkentin and Wang, unpublished results). In this report, we achieved for the first time targeted activation of JNK in adult transgenic hearts using a cre-loxP-mediated, inducible transgenic approach. Analysis of this animal model provided the first in vivo evidence to suggest that JNK activation is sufficient to induce cardiomyopathy in an adult heart, thus implicating a potential role for the JNK pathway in the development of heart failure. Further studies in this transgenic model will help to uncover specific effects of JNK activation on cardiac function and remodeling in fully developed myocardium and to contribute to a better understanding of the molecular/signaling mechanisms of heart failure.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0438fje; to cite this article, use FASEB J. (February 19, 2003) 10.1096/fj.02-0438fje 
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