Regulation of Ncx1 Expression

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. 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. The Na+-Ca2+ exchanger (NCX) 4The abbreviations used are: NCX, Na+-Ca+ exchanger; SRF, serum response factor; ANF, atrial natriuretic factor; m.o.i., multiplicity of infection; GFP, green fluorescent protein; PE, phenylephrine; TAC, transverse aortic constriction; LV, left ventricle; BW, body weight; E, embryonic day. 4The abbreviations used are: NCX, Na+-Ca+ exchanger; SRF, serum response factor; ANF, atrial natriuretic factor; m.o.i., multiplicity of infection; GFP, green fluorescent protein; PE, phenylephrine; TAC, transverse aortic constriction; LV, left ventricle; BW, body weight; E, embryonic day. is one of the essential regulators of Ca2+ homeostasis within cardiomyocytes and is an important regulator of contractility. The exchanger catalyzes the electrogenic exchange of Ca2+ and Na+ across the plasma membrane in either the Ca2+-influx or Ca2+-efflux mode. The NCX family includes three mammalian exchanger genes (Ncx1, Ncx2, and Ncx3) with very similar functional properties (1Linck B. Qiu Z. He Z. Tong Q. Hilgemann D.W. Philipson K.D. Am. J. Physiol. 1998; 274: C415-C423Crossref PubMed Google Scholar). Ncx1 is highly expressed in the heart and there is a rapid up-regulation of Ncx1 in response to pressure overload (2Kent R.L. Rozich J.D. McCollam P.L. McDermott D.E. Thacker U.F. Menick D.R. McDermott P.J. Cooper G.T. Am. J. Physiol. 1993; 265: H1024-H1029Crossref PubMed Google Scholar, 3Muller J.G. Isomatsu Y. Koushik S.V. O'Quinn M. Xu L. Kappler C.S. Hapke E. Zile M.R. Conway S.J. Menick D.R. Circ. Res. 2002; 90: 158-164Crossref PubMed Scopus (29) Google Scholar, 4Wang Z. Nolan B. Kutschke W. Hill J.A. J. Biol. Chem. 2001; 276: 17706-17711Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The exchanger is often found up-regulated in end-stage heart failure and many studies have shown that the change in Ncx1 expression contributes to the pathophysiological phenotype (5Hasenfuss G. Schillinger W. Lehnart S.E. Preuss M. Pieske B. Maier L.S. Prestle J. Minami K. Just H. Circulation. 1999; 99: 641-648Crossref PubMed Scopus (362) Google Scholar, 6Pogwizd S.M. Qi M. Yuan W. Samarel A.M. Bers D.M. Circ. Res. 1999; 85: 1009-1019Crossref PubMed Scopus (358) Google Scholar). Therefore, the up-regulation of the exchanger has been proposed to play an important role in altered excitation-contraction coupling and arrhythmogenesis in the context of cardiac hypertrophy and failure. There are multiple tissue-specific variants of Ncx1 resulting from alternative promoter usage (H1, K1, and Br1) and alternative splicing (3Muller J.G. Isomatsu Y. Koushik S.V. O'Quinn M. Xu L. Kappler C.S. Hapke E. Zile M.R. Conway S.J. Menick D.R. Circ. Res. 2002; 90: 158-164Crossref PubMed Scopus (29) Google Scholar, 7Quednau B.D. Nicoll D.A. Philipson K.D. Am. J. Physiol. 1997; 272: C1250-C1261Crossref PubMed Google Scholar, 8Barnes K.V. Cheng G. Dawson M.M. Menick D.R. J. Biol. Chem. 1997; 272: 11510-11517Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The H1 promoter directs cardiac-specific expression and contains many of the cis elements that have been demonstrated to be important in both regulation of cardiac expression and induction in response to α-adrenergic stimulation. Our previous study in neonatal rat cardiomyocytes showed that a construct containing only 184 bases of the 5′-flanking region, the H1 exon, and 67 bp of the first intron is not only sufficient for cardiac-directed expression but also for α-adrenergic stimulation of the luciferase reporter gene. The mutational analysis revealed that both the CArG box at -80 and the GATA element at -50 were required for expression in rat neonatal cardiomyocyte but were not required for α-adrenergic induction (9Cheng G. Hagen T.P. Dawson M.L. Barnes K.V. Menick D.R. J. Biol. Chem. 1999; 274: 12819-12826Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In contrast to what we found in neonatal cardiomyocytes, the -80 CArG element mediates a part of the α-adrenergic stimulated up-regulation and is required for Ncx1 up-regulation in response to p38 stimulation in isolated adult cardiomyocytes (10Xu L. Kappler C.S. Menick D.R. J. Mol. Cell Cardiol. 2005; 38: 735-743Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). In the present study we examine regulation of exchanger expression in the adult heart. We first characterized the role that each of the cis elements plays in Ncx1 expression in adult feline cardiomyocytes and demonstrated that both the -80 GATA and -50 CArG were important for expression in adult cardiomyocytes. Both GATA4, which binds to GATA elements, and the serum response factor (SRF), which binds to the CArG box (CC(A/T)6GG), play essential roles in cardiac development and regulating hypertrophic growth in the adult heart. Numerous cardiac-expressed genes in addition to the exchanger are regulated by both GATA4 and SRF including ANF, brain natriuretic peptide, α-myosin heavy chain, and β-myosin heavy chain. SRF has been demonstrated to stimulate the expression of cardiac muscle genes in association with a variety of cofactors including LIM domain proteins (11Chang D.F. Belaguli N.S. Iyer D. Roberts W.B. Wu S.P. Dong X.R. Marx J.G. Moore M.S. Beckerle M.C. Majesky M.W. Schwartz R.J. Dev. Cell. 2003; 4: 107-118Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar) and with transcription factors such as GATA4 and Nkx2.5 (12Chen C.Y. Schwartz R.J. Mol. Cell. Biol. 1996; 16: 6372-6384Crossref PubMed Google Scholar, 13Sepulveda J.L. Vlahopoulos S. Iyer D. Belaguli N. Schwartz R.J. J. Biol. Chem. 2002; 277: 25775-25782Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 14Muller J.G. Thompson J.T. Edmonson A.M. Rackley M.S. Kasahara H. Izumo S. McQuinn T.C. Menick D.R. O'Brien T.X. J. Mol. Cell Cardiol. 2002; 34: 807-821Abstract Full Text PDF PubMed Scopus (24) Google Scholar). GATA4 has been shown to mediate inducible expression of several cardiac genes both in in vitro studies in response to phenylephrine, isoproterenol, and endothelin-1 (15Hasegawa K. Lee S.J. Jobe S.M. Markham B.E. Kitsis R.N. Circulation. 1997; 96: 3943-3953Crossref PubMed Scopus (154) Google Scholar, 16Morimoto T. Hasegawa K. Kaburagi S. Kakita T. Wada H. Yanazume T. Sasayama S. J. Biol. Chem. 2000; 275: 13721-13726Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) and in vivo studies in response to pressure overload (17Herzig T.C. Jobe S.M. Aoki H. Molkentin J.D. Cowley Jr., A.W. Izumo S. Markham B.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7543-7548Crossref PubMed Scopus (179) Google Scholar, 18Liang Q. De Windt L.J. Witt S.A. Kimball T.R. Markham B.E. Molkentin J.D. J. Biol. Chem. 2001; 276: 30245-30253Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). The GATA4 and SRF consensus binding regions shown to be important in Ncx1 expression were further characterized utilizing transgenic mouse lines containing reporter genes driven by mutant Ncx1 promoters to determine the role they play in Ncx1 expression in cardiac development and in response to pressure overload in the adult heart. The -50 GATA mutant promoter is still able to drive transgenic expression in both development and in the adult heart and is still capable of being responsive to pressure overload induced up-regulation but the loss of the -50 GATA element results in some non-cardiac-restricted expression of the Ncx1 promoter. The -80 CArG element is critical for transgene expression and response to pressure overload. Lastly, the Ncx1 minimal (184 bp) promoter containing both the -80 CArG and -50 GATA elements is sufficient for driving the normal spatiotemporal pattern of Ncx1 expression in cardiac development but is not sufficient for pressure overload induced up-regulation indicating that at least one additional element is required for hypertrophic regulation of the exchanger. Rat Neonatal and Feline Adult Cardiomyocytes Isolation and Culture—Primary neonatal cardiomyocytes were obtained from 2-4-day-old neonatal rats and cultured by the method described previously (9Cheng G. Hagen T.P. Dawson M.L. Barnes K.V. Menick D.R. J. Biol. Chem. 1999; 274: 12819-12826Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Adult feline cardiomyocytes were isolated via a hanging heart preparation using enzymatic digestion and cultured by the protocols described previously (19Kent R.L. Mann D.L. Urabe Y. Hisano R. Hewett K.W. Loughnane M. Cooper G.T. Am. J. Physiol. 1989; 257: H1717-H1727PubMed Google Scholar). Construction of Adenovirus—Adenoviral constructs were made using the AdEasy system (Stratagene). The Ncx1 promoter-luciferase constructs were cloned into the promoterless pAdTrack vector as described (10Xu L. Kappler C.S. Menick D.R. J. Mol. Cell Cardiol. 2005; 38: 735-743Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Viruses were plaque purified, amplified, and titers determined by the Gazes Adenoviral Core. Cardiomyocytes were infected on day 1 in culture by adding titered adenovirus to the culture medium at a different multiplicity of infection (m.o.i.). After an infection of 8 h the media was changed. When more than one adenoviral construct was used to infect cells, experiments were carried out to ensure there was no competition for infection between the constructs at the multiplicity of infections used. Adult and neonatal cardiomyocytes infected with m.o.i.s of 1 resulted in the infection and gene transfer to greater than 85% of the plated cells based on GFP expression. Chromatin Immunoprecipitation Assay—Adult cardiomyocytes were stimulated with 10 mm phenylephrine (PE) and/or infected with GATA4 or SRF adenovirus (m.o.i. = 1). Forty-eight hours after infection and/or stimulation, cells were treated with 1% (v/v) formaldehyde for 20 min at room temperature with slow rocking. Chromatin immunoprecipitation assay was performed as described in the manufacturer's manual (Upstate) with some modifications. Cells were washed two times with ice-cold phosphate-buffered saline and collected by centrifugation at 10,000 × g for 2 min. The cell pellet was suspended in lysis buffer and incubated in ice for 20 min. The cell lysate was sonicated 10 times for 10 s each and the cell debris spun down. The sample was pre-cleared and the immunoprecipitation antibody added to the supernatant and incubated overnight at 4 °C. After immunoprecipitation, the eluted protein-DNA complexes were de-cross-linked by heating at 65 °C for 4 h. The DNA was ethanol precipitated and the DNA was suspended in 50 μl of 10 mm Tris buffer. The feline Ncx1 proximal promoter was PCR amplified from the immunoprecipitated and non-immunoprecipitated chromatin using the following primers: sense -152 to -131 (5′-GTGTTGGATGAAGCGGAGAG-3′) and antisense -14 to -34 (5′-AACATGGTTTGCATAGCTGCA-3′). Production of Transgenic Mouse Lines—The Ncx1 promoter-driven luciferase transgenic mice were produced as described previously (3Muller J.G. Isomatsu Y. Koushik S.V. O'Quinn M. Xu L. Kappler C.S. Hapke E. Zile M.R. Conway S.J. Menick D.R. Circ. Res. 2002; 90: 158-164Crossref PubMed Scopus (29) Google Scholar). Briefly, the 1831-bp full-length feline Ncx1 promoter construct or its mutants (-50 GATA and -80 CarG) were introduced into the multiple cloning site of the luciferase reporter vector pGL2 (Promega, Madison, WI). The 184Ncx1 promoter construct contained the first 184 bases of the feline promoter, the H1 exon, and 67 bases of the first intron fused to the β-galactosidase reporter gene. The resulting constructs were digested, and gel-purified. Pronuclear microinjection in the FVB/N mouse strain was performed at the Transgenic Core Facility of the Medical University of South Carolina. Founder mice were identified by PCR and confirmed by genomic Southern blots. Multitransgenic lines for each construct were maintained by mating transgenic offspring with normal FVB/N mice from Taconic Labs (Germantown, NY). Histological Analysis—Embryo dissection, tissue isolation, fixation, and processing for lacZ staining (n = 23) were carried out as described (20Koushik S.V. Wang J. Rogers R. Moskophidis D. Lambert N.A. Creazzo T.L. Conway S.J. FASEB J. 2001; 15: 1209-1211Crossref PubMed Scopus (217) Google Scholar). Similarly, all lacZ stained embryonic, newborn and adult samples were then dehydrated, embedded in paraffin, and sectioned at 6 μm thickness for hematoxylin and eosin staining and microscopic analysis according to standard procedures (20Koushik S.V. Wang J. Rogers R. Moskophidis D. Lambert N.A. Creazzo T.L. Conway S.J. FASEB J. 2001; 15: 1209-1211Crossref PubMed Scopus (217) Google Scholar). In situ hybridization was carried out as described (3Muller J.G. Isomatsu Y. Koushik S.V. O'Quinn M. Xu L. Kappler C.S. Hapke E. Zile M.R. Conway S.J. Menick D.R. Circ. Res. 2002; 90: 158-164Crossref PubMed Scopus (29) Google Scholar). Pressure Overload Mouse Model—Left ventricular pressure overload was created by microsurgical transverse aortic constriction (TAC) as described previously (3Muller J.G. Isomatsu Y. Koushik S.V. O'Quinn M. Xu L. Kappler C.S. Hapke E. Zile M.R. Conway S.J. Menick D.R. Circ. Res. 2002; 90: 158-164Crossref PubMed Scopus (29) Google Scholar). Briefly, transgenic FVB/N mice carrying the Ncx1H1-luc construct or mutants were anesthetized with ketamine (50 mg/kg) and xylazine (2.5 mg/kg) and respiration was artificially controlled. The transverse aorta was constricted by tying a suture around the vessel over a 28-gauge or a 30-gauge blunted needle causing complete occlusion of the aorta. The needle was withdrawn, resulting in a severely stenotic aortic lumen. Two and 7 days after surgery, animals were sacrificed by removal of the heart in deep anesthesia. Echocardiography and Doppler echocardiography were performed on mice before banding (baseline echo) and just prior to sacrifice (final echo). A 15-MHz transducer (Sonos 5500, Agilent Technologies, Andover, MA) was placed on a layer of acoustic coupling gel applied to the hemithorax. Measurements of LV dimension and posterior wall thickness were made at end systole and end diastole according to the leading edge convention of the American Society of Echocardiography. Three to six beats were averaged for each measurement. Fractional shortening and LV mass were calculated as we have described previously (21Roten L. Nemoto S. Simsic J. Coker M.L. Rao V. Baicu S. Defreyte G. Soloway P.J. Zile M.R. Spinale F.G. J. Mol. Cell Cardiol. 2000; 32: 109-120Abstract Full Text PDF PubMed Scopus (106) Google Scholar) with standard equations. Standard Doppler techniques were used to measure the velocity (V) across the transverse aortic constriction. Maximum peak pressure gradient was then calculated using a modified Bernoulli equation as 4V2. Left and right ventricular weights were determined after microdissection and tissue samples of the heart and other organs were snap-frozen in liquid nitrogen. The mouse body weight (BW) at the end of the study was used for indexing purposes. All animal experimentation was performed in accordance with National Institutes of Health Guidelines, and protocols were approved by the Animal Care Committee of the Medical University of South Carolina. Luciferase and β-Galactosidase Activity and GFP Assays—To quantitate Ncx1-promoter reporter gene expression, samples of heart tissue were ground in a mortar under liquid nitrogen and assayed for luciferase activity as previously described (3Muller J.G. Isomatsu Y. Koushik S.V. O'Quinn M. Xu L. Kappler C.S. Hapke E. Zile M.R. Conway S.J. Menick D.R. Circ. Res. 2002; 90: 158-164Crossref PubMed Scopus (29) Google Scholar). β-Galactosidase activity was measured using the Galacto-Light and Galacto-Light Plus Systems (AB Applied Biosystems, Foster City, CA) following the manufacturer's protocol. Luciferase activity and GFP fluorescence from adenoviral infected neonatal and adult cardiomyocytes were assayed as described (10Xu L. Kappler C.S. Menick D.R. J. Mol. Cell Cardiol. 2005; 38: 735-743Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Statistical Analyses—Multiple comparisons of experimental groups were done with factorial analysis of variance and the Scheffe F post hoc test. A value of p < 0.05 was considered to be significant. All data are shown as mean ± S.E. The 184Ncx1 Minimal Promoter Is Sufficient for Expression in Adult Cardiomyocytes but Not for Up-regulation—Fig. 1 demonstrates that the Ncx1 minimal promoter is sufficient to direct expression of the luciferase reporter gene in both neonatal and adult cardiomyocytes. The 184Ncx1 promoter drives reporter gene expression at levels 3-4-fold greater than the full-length Ncx1 (1831Ncx1) promoter in both neonatal and adult cardiomyocytes (Fig. 1, A and B) because of the deletion of putative repressor elements distal to the minimal 184-bp promoter construct. 5L. Xu and D. R. Menick, unpublished data. The 1831Ncx1 promoter is up-regulated in response to α-adrenergic stimulation in both adult and neonatal cardiomyocytes. However, the 184Ncx1 promoter is up-regulated in response to α-adrenergic stimulation in neonatal cardiomyocytes but is recalcitrant to α-adrenergic stimulation in adult cardiomyocytes (compare Fig. 1, A to B). These data demonstrate that α-adrenergic-stimulated up-regulation of Ncx1 is different in the neonatal cardiomyocyte when compared with the adult cell. To test whether the Ncx1 minimal promoter contains sufficient DNA regulatory elements to direct cardiac-specific expression, we established two independent 184Ncx1-β-galactosidase transgenic mouse lines. Moderate levels of β-galactosidase were detected in the hearts, and only the hearts, of both lines expressing the transgene. The 184Ncx1-driven β-galactosidase expression was initially weakly detectable in E8.5 embryos (not shown) and robustly detected in E9.5 (Fig. 2A) embryos. Significantly, heart-restricted lacZ expression was present in 100% of the transgenic embryos (n = 14) and was absent from all the non-transgenic embryos examined (n = 31). When compared with the endogenous Ncx1 mRNA and Ncx1lacZ knock-in reporter expression patterns (20Koushik S.V. Wang J. Rogers R. Moskophidis D. Lambert N.A. Creazzo T.L. Conway S.J. FASEB J. 2001; 15: 1209-1211Crossref PubMed Scopus (217) Google Scholar, 22Koushik S.V. Bundy J. Conway S.J. Mech. Dev. 1999; 88: 119-122Crossref PubMed Scopus (30) Google Scholar), the lacZ spatiotemporal patterns were very similar (Fig. 2B). Histological sectioning revealed that both 184Ncx1 and Ncx1lacZ lacZ expression was restricted to the cardiomyocytes and absent from the adjacent endothelial and endocardial cushion cells (not shown). However, when older E14 (not shown) and newborn (Fig. 2, C and D) hearts were examined, a less prevalent and “patchy” lacZ expression was detected within subpopulations of the cardiomyocytes within the ventricles and atria of both transgenic lines, when compared with the endogenous Ncx1 mRNA (23Conway S.J. Kruzynska-Frejtag A. Wang J. Rogers R. Kneer P.L. Chen H. Creazzo T. Menick D.R. Koushik S.V. Ann. N. Y. Acad. Sci. 2002; 976: 268-281Crossref PubMed Scopus (15) Google Scholar) and Ncx1lacZ knock-in reporter expression patterns (20Koushik S.V. Wang J. Rogers R. Moskophidis D. Lambert N.A. Creazzo T.L. Conway S.J. FASEB J. 2001; 15: 1209-1211Crossref PubMed Scopus (217) Google Scholar). The decreased prevalence and mosaic pattern of expression observed at E14.5 (not shown) and newborn stages persisted into the adult (data not shown). Additionally in these later stages, lacZ reporter activity could be detected in the sinus venosus, pulmonary and caval vein region (Fig. 2D). Importantly, no β-galactosidase activity was detected in the kidney, liver, spleen, brain, or skeletal muscle of the embryonic, newborn, or adult transgenic animals (Table 1). These data reveal that the 184Ncx1 minimal promoter retains the necessary enhancer elements to drive initial early cardiomyocyte-specific reporter expression, but that subsequently undefined enhancer elements required for ubiquitous cardiomyocyte lacZ reporter activity have been deleted.TABLE 1Tissue distribution of luciferase activity in adult 1831Ncx1, 1831Ncx1 GATA -50 mutant and 1831Ncx1 CArG -80 mutant, and β-galactosidase activity in 184Ncx1 transgenic miceLuciferase activityTissue1831Ncx1Ncx1 –50 GATANcx1 –80 CArGβ-Galactosidase activity,aAll values shown are for the luciferase of β-galactosidase activity of the transgenic mouse tissue minus background value of same tissue in non-transgenic mouse 184Ncx1RLUbRLU, relative light unitsRLULeft ventricle37,96420036.71899Brain0000Kidney017.13.90Lung09.500Liver0000Spleen0000Skeletal muscle037.56.20Uterus06000Aorta015.200a All values shown are for the luciferase of β-galactosidase activity of the transgenic mouse tissue minus background value of same tissue in non-transgenic mouseb RLU, relative light units Open table in a new tab Ncx1 transcript and protein levels are increased in response to pressure overload in the feline model of hypertrophy (2Kent R.L. Rozich J.D. McCollam P.L. McDermott D.E. Thacker U.F. Menick D.R. McDermott P.J. Cooper G.T. Am. J. Physiol. 1993; 265: H1024-H1029Crossref PubMed Google Scholar) and this up-regulation is mediated by the Ncx1 H1 promoter (3Muller J.G. Isomatsu Y. Koushik S.V. O'Quinn M. Xu L. Kappler C.S. Hapke E. Zile M.R. Conway S.J. Menick D.R. Circ. Res. 2002; 90: 158-164Crossref PubMed Scopus (29) Google Scholar). To determine whether the minimal Ncx1 promoter is sufficient for the up-regulation of the exchanger in response to pressure overload hypertrophy, transgenic mice expressing either the 184Ncx1-β-galactosidase, or the wild-type 1831Ncx1-luciferase were subjected to cardiac LV pressure overload by microsurgical TAC. Sham-operated littermates underwent the same anesthesia and thoracotomy protocol without TAC to control for the effect of anesthesia and surgical trauma. The mice from both sham-operated and TAC groups from each transgenic line were euthanized 2 and 7 days after surgery. TAC with either the 28- or 30-gauge needles resulted in a pressure gradient greater than 90 mm Hg. However, 7 days after TAC, LV/BW increased by 31% using a 28-gauge needle and increased by 81% using a 30-gauge needle (Fig. 3, A and B). Protein extracts were prepared from LV tissue samples from control, sham-operated, and TAC-operated transgenic mice to quantify the response of the wild-type and mutant Ncx1-promoter reporter transgene. Reporter gene activity was assayed and normalized for protein content. In the 1831Ncx1 mice, the 28-gauge was sufficient to induce a significant hypertrophic stimulus and increase in luciferase activity. As previously reported (3Muller J.G. Isomatsu Y. Koushik S.V. O'Quinn M. Xu L. Kappler C.S. Hapke E. Zile M.R. Conway S.J. Menick D.R. Circ. Res. 2002; 90: 158-164Crossref PubMed Scopus (29) Google Scholar), endogenous Ncx1 protein levels are up-regulated after 7 days of pressure overload (not shown) and the wild-type full-length 1831Ncx1 promoter driven reporter gene activity showed a 2-fold up-regulation after 7 days of pressure overload (Fig. 3C). Preliminary studies in the 184Ncx1 mice did not produce an increase in β-galactosidase reporter activity. To be certain that this was not a false negative response (i.e. insufficient hypertrophic stimulus), we used a 30-gauge needle for TAC in the 184Ncx1 mice. Despite the robust increase in LV mass (81% increase in LV/BW) after 7 days of TAC, there was no up-regulation of the 184Ncx1-β-galactosidase activity when compared with controls after 7 days of TAC (Fig. 3D). Clearly one or more cis elements responsible for Ncx1 up-regulation are distal to the minimal 184-bp promoter construct. Role of -50 GATA and -80 CArG Elements in the Regulation of Ncx1—The NCX1 minimal promoter contains several consensus sequences for a number of potential DNA binding factors that have been shown to be important in regulating NCX1 expression in neonatal cardiomyocytes and have also been shown to be important in regulating the cardiac expression of ANF, brain natriuretic peptide, β-myosin heavy chain, and skeletal α-actin (24Sepulveda J.L. Belaguli N. Nigam V. Chen C.Y. Nemer M. Schwartz R.J. Mol. Cell. Biol. 1998; 18: 3405-3415Crossref PubMed Scopus (276) Google Scholar). These include two GATA elements, two E-Box elements, a CArG element, and a binding site for Nkx 2.5 (Fig. 4). It is important to note that the first 184 bp of the human (25Kraev A. Chumakov I. Carafoli E. Genomics. 1996; 37: 105-112Crossref PubMed Scopus (44) Google Scholar) and feline (8Barnes K.V. Cheng G. Dawson M.M. Menick D.R. J. Biol. Chem. 1997; 272: 11510-11517Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) Ncx1 H1 promoters have 97% identity and the rat (26Scheller T. Kraev A. Skinner S. Carafoli E. J. Biol. Chem. 1998; 273: 7643-7649Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) and feline sequences have 92% identity. Using the 1831Ncx1 construct, we introduced site-specific point mutations into each of these elements, and the activity in adult cardiomyocytes of each of these adenoviral constructs was compared with the wild-type construct to determine its contribution to expression. Ablation of the -172 E-Box, -153 E-Box, -10 NKE, and the novel element at +103 resulted in >50% of the reporter activity seen in the wild-type promoter-luciferase construct (Fig. 5A). Mutation of the -123 GATA or -50 GATA element resulted in 35 and 29% of wild-type promoter activity, respectively. A point mutation disrupting the -80 CArG element resulted in luciferase activity of less than 20% of the control level. Clearly the CArG and GATA elements are important to basal Ncx1 expression in adult cardiomyocytes.FIGURE 5Effect of Ncx1 promoter transcriptional element mutations on basal and adrenergic stimulation of NCX1 promoter-driven reporter gene expression in adult feline cardiomyocytes. A, adult feline cardiomyocytes were infected with the wild-type or mutant Ncx1 promoter-luciferase reporter adenoviruses (m.o.i. 1.5). Cells were then incubated for 48 h and lysed in reporter buffer. Luciferase activity was dete