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J. Biol. Chem., Vol. 281, Issue 45, 34430-34440, November 10, 2006

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Regulation of Ncx1 Expression

IDENTIFICATION OF REGULATORY ELEMENTS MEDIATING CARDIAC-SPECIFIC EXPRESSION AND UP-REGULATION ^*

Lin Xu, Ludivine Renaud, Joachim G. Müller¹, Catalin F. Baicu, D. Dirk Bonnema, Hongming Zhou, Christiana S. Kappler², Steven W. Kubalak, Michael R. Zile, Simon J. Conway, and Donald R. Menick³

From the Gazes Cardiac Research Institute, Division of Cardiology, Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425 and the Cardiovascular Development Group, Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana 46202

The Na⁺-Ca²⁺ exchanger (NCX1) is up-regulated in hypertrophy and is often found up-regulated in end-stage heart failure. Studies have shown that the change in its expression contributes to contractile dysfunction. We have previously shown that the 1831-bp Ncx1 H1 (1831Ncx1) promoter directs cardiac-specific expression of the exchanger in both development and in the adult, and is sufficient for the up-regulation of Ncx1 in response to pressure overload. Here, we utilized adenoviral mediated gene transfer and transgenics to identify minimal regions and response elements that mediate Ncx1 expression in the heart. We demonstrate that the proximal 184 bp of the Ncx1 H1 (184Ncx1) promoter is sufficient for expression of reporter genes in adult cardiomyocytes and for the correct spatiotemporal pattern of Ncx1 expression in development but not for up-regulation in response to pressure overload. Mutational analysis revealed that both the -80 CArG and the -50 GATA elements were required for expression in isolated adult cardiomyocytes. Chromatin immunoprecipitation assays in adult cardiocytes demonstrate that SRF and GATA4 are associated with the proximal region of the endogenous Ncx1 promoter. Transgenic lines were established for the 1831Ncx1 promoter-luciferase containing mutations in the -80 CArG or -50 GATA element. No luciferase activity was detected during development, in the adult, or after pressure overload in any of the -80 CArG transgenic lines. The Ncx1 -50 GATA mutant promoter was sufficient for driving the normal spatiotemporal pattern of Ncx1 expression in development and for up-regulation in response to pressure overload but importantly, expression was no longer cardiac restricted. This work is the first in vivo study that demonstrates which cis elements are important for Ncx1 regulation.

Received for publication, August 24, 2006

^* This work was supported in part by National Institutes of Health Grants HL066223 and P01 HL48788 (project 3) (to D. R. M.) and HL60714 (to S. J. C.) and NCRR16434 (To L. X.) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by Deutsche Forschungsgemeinschaft Grant Mu 1454/1-1. Current address: Dept. of Medicine, Fletcher Allen Health Care, University of Vermont, Burlington, VT 05401.

² Supported by National Institutes of Health Predoctoral Fellowship T32HL07260.

³ To whom correspondence should be addressed: 114 Doughty St. Charleston, SC 29425. Tel.: 843-876-5045; E-mail: menickd{at}musc.edu.

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