The cardiac Na ؉ -Ca 2؉ exchanger (NCX1) is the principal Ca 2؉ efflux mechanism in cardiocytes. The exchanger is up-regulated in both cardiac hypertrophy and failure. In this report, we identify the cis-acting elements that control cardiac expression and ␣-adrenergic up-regulation of the exchanger gene. Deletion analysis revealed that a minimal cardiac promoter fragment from ؊184 to ؉172 is sufficient for cardiac expression and ␣-adrenergic stimulation. Mutational analysis revealed that both the CArG element at ؊80 and the GATA element at ؊50 were required for cardiac expression. Gel mobility shift assay supershift analysis demonstrated that the serum response factor binds to the CArG element and GATA-4 binds to the GATA element. Point mutations in the ؊172 E-box demonstrated that it was required for ␣-adrenergic induction. In addition, deletion analysis revealed one or more enhancer elements in the first intron (؉103 to ؉134) that are essential for phenylephrine up-regulation but bear no homology to any known transcription element. Therefore, this work demonstrates that SRF and GATA-4 are critical for NCX1 expression in neonatal cardiomyocytes and that the ؊172 E-box in addition to a novel enhancer element(s) are required for phenylephrine up-regulation of NCX1 and may mediate its hypertrophic up-regulation.The Na ϩ -Ca 2ϩ exchanger (NCX1) 1 catalyzes the electrogenic exchange of one intracellular calcium ion for three extracellular sodium ions across the plasma membrane in many mammalian cells. Transport is reversible and can facilitate calcium entry, which in the heart is capable of triggering calcium release from the sarcoplasmic reticulum (1). The exchanger is most abundant in the heart, where it regulates Ca 2ϩ fluxes across the sarcolemma and serves a critical role in the maintenance of the cellular calcium balance for excitation-contraction coupling. Na ϩ -Ca 2ϩ exchanger activity in cardiomyocytes is regulated by several factors. It is activated by cytosolic Ca 2ϩ and MgATP (2) and inhibited by cytosolic sodium (3) and ATP depletion (4). A high affinity Ca 2ϩ -binding domain has been identified in the large cytoplasmic loop (residues 371-508) that is believed to be responsible for calcium regulation (5). It is also inhibited by the exchanger inhibitory peptide, which corresponds to a 20-amino acid segment at the N terminus of the large cytoplasmic loop (6). A recent study has demonstrated that the exchanger is phosphorylated via a protein kinase C-dependent pathway and that NCX1 phosphorylation appears to coincide with up-regulation of exchanger activity (7).In addition, the exchanger is regulated at the transcriptional level in cardiac hypertrophy, ischemia, and failure. In the feline model of acute right ventricular hypertrophy, NCX1 message levels are rapidly up-regulated following pressure overload (8,9). An increase in NCX1 mRNA expression is also observed in cultured cardiac myocytes following ␣-adrenergic stimulation by phenylephrine or exposure to veratridine. Importantly, the exchanger is also...

The Na ؉-Ca 2؉ exchanger (NCX1) plays a major role in calcium efflux and therefore in the control and regulation of intracellular calcium in the heart. The exchanger has been shown to be regulated at several levels including transcription. NCX1 mRNA levels are up-regulated in both cardiac hypertrophy and failure. In this work, the 5-end of the ncx1 gene has been cloned to study the mechanisms that mediate hypertrophic stimulation and cardiac expression. The feline ncx1 gene has three exons that encode 5-untranslated sequences that are under the control of three tissue-specific promoters. The cardiac promoter drives expression in cardiocytes, but not in mouse L cells. Although it contains at least one enhancer (؊2000 to ؊1250 base pairs (bp)) and one or more negative elements (؊1250 to ؊250 bp), a minimum promoter (؊250 to ؉200 bp) is sufficient for cardiac expression and ␣-adrenergic stimulation.

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