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• W2000398093 abstract "The cardiac Na+/Ca2+ exchanger (NCX1) is the predominant mechanism for the extrusion of Ca2+ from beating cardiomyocytes. The role of protein phosphorylation in the regulation of NCX1 function in normal and diseased hearts remains unclear. In our search for proteins that interact with NCX1 using a yeast two-hybrid screen, we found that the C terminus of calcineurin Aβ, containing the autoinhibitory domain, binds to the β1 repeat of the central cytoplasmic loop of NCX1 that presumably constitutes part of the allosteric Ca2+ regulatory site. The association of NCX1 with calcineurin was significantly increased in the BIO14.6 cardiomyopathic hamster heart compared with that in the normal control. In hypertrophic neonatal rat cardiomyocytes subjected to chronic phenylephrine treatment, we observed a marked depression of NCX activity measured as the rate of Na+i-dependent 45Ca2+ uptake or the rate of Na+o-dependent 45Ca2+ efflux. Depressed NCX activity was partially and independently reversed by the acute inhibition of calcineurin and protein kinase C activities with little effect on myocyte hypertrophic phenotypes. Studies of NCX1 deletion mutants expressed in CCL39 cells were consistent with the view that the β1 repeat is required for the action of endogenous calcineurin and that the large cytoplasmic loop may be required to maintain the interaction of the enzyme with its substrate. Our data suggest that NCX1 is a novel regulatory target for calcineurin and that depressed NCX activity might contribute to the etiology of in vivo cardiac hypertrophy and dysfunction occurring under conditions in which both calcineurin and protein kinase C are chronically activated. The cardiac Na+/Ca2+ exchanger (NCX1) is the predominant mechanism for the extrusion of Ca2+ from beating cardiomyocytes. The role of protein phosphorylation in the regulation of NCX1 function in normal and diseased hearts remains unclear. In our search for proteins that interact with NCX1 using a yeast two-hybrid screen, we found that the C terminus of calcineurin Aβ, containing the autoinhibitory domain, binds to the β1 repeat of the central cytoplasmic loop of NCX1 that presumably constitutes part of the allosteric Ca2+ regulatory site. The association of NCX1 with calcineurin was significantly increased in the BIO14.6 cardiomyopathic hamster heart compared with that in the normal control. In hypertrophic neonatal rat cardiomyocytes subjected to chronic phenylephrine treatment, we observed a marked depression of NCX activity measured as the rate of Na+i-dependent 45Ca2+ uptake or the rate of Na+o-dependent 45Ca2+ efflux. Depressed NCX activity was partially and independently reversed by the acute inhibition of calcineurin and protein kinase C activities with little effect on myocyte hypertrophic phenotypes. Studies of NCX1 deletion mutants expressed in CCL39 cells were consistent with the view that the β1 repeat is required for the action of endogenous calcineurin and that the large cytoplasmic loop may be required to maintain the interaction of the enzyme with its substrate. Our data suggest that NCX1 is a novel regulatory target for calcineurin and that depressed NCX activity might contribute to the etiology of in vivo cardiac hypertrophy and dysfunction occurring under conditions in which both calcineurin and protein kinase C are chronically activated. The Na+/Ca2+ exchanger (NCX) 1The abbreviations used are: NCX, Na+/Ca2+ exchanger; NCX1, cardiac isoform of NCX; aa, amino acids; ANP, atrial natriuretic peptide; BSS, balanced salt solution; CnA, calcineurin A; DOX, doxycycline; FCS, fetal calf serum; PE, phenylephrine; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; Rp-8-CPT-cAMPS, 8-(4-chlorophenylthio)adenosine-3′,5′-cyclic monophosphorothioate; Rp-8-CPT-cGMPS, 8-(4-chlorophenylthio)guanosine-3′,5′-cyclic monophosphorothioate; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling.1The abbreviations used are: NCX, Na+/Ca2+ exchanger; NCX1, cardiac isoform of NCX; aa, amino acids; ANP, atrial natriuretic peptide; BSS, balanced salt solution; CnA, calcineurin A; DOX, doxycycline; FCS, fetal calf serum; PE, phenylephrine; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; Rp-8-CPT-cAMPS, 8-(4-chlorophenylthio)adenosine-3′,5′-cyclic monophosphorothioate; Rp-8-CPT-cGMPS, 8-(4-chlorophenylthio)guanosine-3′,5′-cyclic monophosphorothioate; TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling. catalyzes the reversible exchange of Na+ for Ca2+ across the plasma membrane. In normal cardiac muscle, the primary role of NCX1 (the cardiac isoform of NCX) is to extrude cytoplasmic Ca2+ during myocyte repolarization and diastole, which balances Ca2+ entry via L-type Ca2+ channels during myocyte depolarization (1Bridge J.H.B. Smolley J.R. Spitzer K.W. Science. 1990; 248: 376-378Crossref PubMed Scopus (228) Google Scholar, 2Bers D.M. Circ. Res. 2000; 87: 275-281Crossref PubMed Scopus (465) Google Scholar). The transport activity of NCX1 is known to be influenced by a variety of factors, including hormones and growth factors, intracellular Na+ and Ca2+ concentrations, membrane potential, cytoplasmic ATP, and protein and lipid phosphorylation (3Shigekawa M. Iwamoto T. Circ. Res. 2001; 88: 864-876Crossref PubMed Scopus (208) Google Scholar). However, information is still limited as to the quantitative aspects of changes in these factors and their consequences on NCX1 activity in normal and diseased cardiomyocytes. For example, in hypertrophic and failing hearts from human patients and animal models, sarcolemmal NCX1 expression has often been shown to be elevated (4Kent R.L. Rozich J.D. McCollam P.L. McDemott D.E. Thacker U.F. Menick D.R. McDermott P.J. Cooper IV, G. Am. J. Physiol. 1993; 265: H1024-H1029Crossref PubMed Google Scholar, 5Studer R. Reinecke H. Biger J. Eschenhagen T. Bo ̈hm M. Hasenfuss G. Just H. Holtz J. Drexler H. Circ. Res. 1994; 75: 443-453Crossref PubMed Scopus (517) Google Scholar, 6Ahmmed G.U. Dong P.E. Song G. Ball N.A. Xu Y. Walsh R.A. Chiamvimonvat N. Circ. Res. 2000; 86: 558-570Crossref PubMed Scopus (85) Google Scholar, 7O'Rourke B. Kass D.A. Tomaselli G.F. Ka ̈a ̈b S. Tunin R. Marba ́n E. Circ. Res. 1999; 6: 558-570Google Scholar), which could be compensatory for the reduced ability of the sarcoplasmic reticulum to maintain low diastolic [Ca2+]i under these pathological conditions. However, whether increased NCX expression invariably leads to enhanced function under disease conditions is not clear, although enhanced NCX expression and function have been observed in cardiomyocytes isolated from some animal models of cardiac hypertrophy and heart failure (6Ahmmed G.U. Dong P.E. Song G. Ball N.A. Xu Y. Walsh R.A. Chiamvimonvat N. Circ. Res. 2000; 86: 558-570Crossref PubMed Scopus (85) Google Scholar, 7O'Rourke B. Kass D.A. Tomaselli G.F. Ka ̈a ̈b S. Tunin R. Marba ́n E. Circ. Res. 1999; 6: 558-570Google Scholar). Protein phosphorylation is an important mechanism regulating the functions of many cellular systems. In the case of NCX1, acute treatment with PMA or agonists of Gαq-coupled receptors such as phenylephrine (PE) has been shown to enhance NCX activity in isolated cardiomyocytes as well as in cells expressing cloned NCX1 (8Iwamoto T. Pan Y. Wakabayashi S. Imagawa T. Yamanaka H.I. Shigekawa M. J. Biol. Chem. 1996; 271: 13609-13615Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 9Iwamoto T. Pan Y. Nakamura T.Y. Wakabayashi S. Shigekawa M. Biochemistry. 1998; 37: 17230-17238Crossref PubMed Scopus (94) Google Scholar, 10Stengl M. Mubagwa K. Carmeliet E. Flameng W. Cardiovasc. Res. 1998; 38: 703-710Crossref PubMed Scopus (40) Google Scholar). Protein kinase A activation has also been reported to stimulate NCX1 activity (11Link B. Qiu Z. He Z. Tong Q. Hilgemann D.W. Philipson K.D. Am. J. Physiol. 1998; 274: C415-C423Crossref PubMed Google Scholar, 12Wei S.-K. Ruknudin A. Hanlon S.U. McCurley J.M. Schulze D.H. Haigney M.C.P. Circ. Res. 2003; 92: 897-903Crossref PubMed Scopus (79) Google Scholar). On the other hand, a protein phosphatase inhibitor, calyculin A, reportedly causes substantial inhibition of NCX activity in cells expressing cloned NCX1 (13Condrescu M. Hantash B.M. Fang Y. Reeves J.P. J. Biol. Chem. 1999; 274: 33279-33286Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). NCX1 stimulation by PMA and agonists of Gαq-coupled receptors occurs via a mechanism involving PKC activation and requires the participation of the central cytoplasmic loop of the exchanger (see Fig. 1a) (9Iwamoto T. Pan Y. Nakamura T.Y. Wakabayashi S. Shigekawa M. Biochemistry. 1998; 37: 17230-17238Crossref PubMed Scopus (94) Google Scholar). Because these agonist effects did not require the direct phosphorylation of NCX1, the central cytoplasmic loop was considered to serve as an anchor for phosphorylatable regulatory ancillary protein(s) (9Iwamoto T. Pan Y. Nakamura T.Y. Wakabayashi S. Shigekawa M. Biochemistry. 1998; 37: 17230-17238Crossref PubMed Scopus (94) Google Scholar). In this study, we undertook a search for regulatory proteins interacting with the central cytoplasmic loop of NCX1 by using a yeast two-hybrid screen. From this search and subsequent analysis, we identified a complex, hitherto unrecognized regulatory mechanism for cardiac NCX1 involving calcineurin and PKC in hypertrophic cardiomyocytes subjected to prolonged PE pretreatment. This mechanism is capable of markedly depressing NCX1 activity. Because calcineurin acts as a central mediator of in vivo cardiac hypertrophy and failure (14Molkentin J.D. Lu J.-R. Antos C.L. Markham B. Richardson J. Robins J. Grant S.R. Olson E.N. Cell. 1998; 93: 215-228Abstract Full Text Full Text PDF PubMed Scopus (2203) Google Scholar, 15Molkentin J.D. Dorn II, G.W. Ann. Rev. Physiol. 2001; 63: 391-426Crossref PubMed Scopus (577) Google Scholar, 16Vega R.B. Bassel-Duby R. Olson E.N. J. Biol. Chem. 2003; 278: 36981-36984Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), NCX might contribute to the etiology of in vivo cardiac dysfunction. Materials—FCS, PE, FK506, PMA, calphostin C, 8-bromo-cAMP, Rp-8-CPT-cAMPS, 8-bromo-cGMP, Rp-8-CPT-cGMPS, and H89 were purchased from Sigma. GF109203X, chelerythrine chloride, KN93, and KN62 were purchased from Calbiochem. Rhodamine-conjugated phalloidin was obtained from Molecular Probes. Antibodies to NCX isoforms have been described (8Iwamoto T. Pan Y. Wakabayashi S. Imagawa T. Yamanaka H.I. Shigekawa M. J. Biol. Chem. 1996; 271: 13609-13615Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 9Iwamoto T. Pan Y. Nakamura T.Y. Wakabayashi S. Shigekawa M. Biochemistry. 1998; 37: 17230-17238Crossref PubMed Scopus (94) Google Scholar). Rabbit polyclonal anti-pan calcineurin A (CnA) and goat polyclonal anti-CnAβ (Santa Cruz Biotechnology), mouse monoclonal anti-CnAβ (Upstate Technology), anti-hemagglutinin (Roche Applied Science), rabbit polyclonal anti-atrial natriuretic peptide (anti-ANP; Phoenix Pharmaceuticals), fluorescein isothiocyanate-conjugated anti-rabbit IgG and rhodamine-conjugated anti-goat IgG (ICN/CAPEL), and anti-calmodulin, horseradish peroxidase-conjugated anti-rabbit IgG, and biotin-conjugated anti-mouse IgG (Zymed Laboratories Inc.) were purchased the from the sources indicated in parentheses. Horseradish peroxidase-conjugated streptavidin was obtained from Zymed Laboratories Inc.. 45CaCl2 was purchased from Amersham Biosciences. Animals—Pregnant Wister rats and male Bio14.6 hamsters (J2N-k strain, 120 days old) and age-matched normal controls (J2N-n) were purchased from Japan SLC. The J2N-n had the same genetic background as the J2N-k, except for the difference of a genetic locus for cardiomyopathy. Cell Cultures—Primary cardiomyocyte cultures were prepared from ventricles of 1-day-old rats as described previously (17Simpson P. McGrath A. Savion S. Circ. Res. 1982; 51: 787-801Crossref PubMed Scopus (373) Google Scholar). They were plated on collagen-coated 24-well dishes at a density of 4×106 cells per well and maintained in M199 medium supplemented with 10% FCS. Staining with rhodamine-phalloidin revealed that >90% of the cells were cardiomyocytes. Two days later, the cells were divided into three groups and then maintained for up to 5 days in M199 alone, M199 with 10 μm PE, or M199 with 10% FCS (see Fig. 3a). These myocytes usually form clusters and exhibit spontaneous synchronized beating (17Simpson P. McGrath A. Savion S. Circ. Res. 1982; 51: 787-801Crossref PubMed Scopus (373) Google Scholar). On the other hand, CCL39 cells (American Type Culture Collection) and their NCX1 transfectants were maintained in Dulbecco's modified Eagle's medium containing 7.5% FCS, 50 units/ml penicillin, and 50 μg/ml streptomycin. Construction of Vectors and Expression of NCX1 and Calcineurin— The preparation of cDNAs of dog heart NCX1 and NCX1 mutants lacking aa 246–672 (NCX1Δ246–672) or aa 407–478 (NCX1Δ407–478), their transfection into CCL39 cells, and the isolation of cell clones stably expressing high NCX activity were carried out as described previously (8Iwamoto T. Pan Y. Wakabayashi S. Imagawa T. Yamanaka H.I. Shigekawa M. J. Biol. Chem. 1996; 271: 13609-13615Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 9Iwamoto T. Pan Y. Nakamura T.Y. Wakabayashi S. Shigekawa M. Biochemistry. 1998; 37: 17230-17238Crossref PubMed Scopus (94) Google Scholar). The isolation of cDNA encoding human CnAβ and the construction of its constitutively active mutant (ΔCnA) lacking the autoinhibitory and the calmodulin binding domains were described previously (18Shibasaki F. MacKeon F. J. Cell Biol. 1995; 131: 735-743Crossref PubMed Scopus (229) Google Scholar, 19Wang H.-G. Pathan N. Ethell I.M. Krajewski S. Yamaguchi Y. Shibasaki F. Mckeon F. Bobo T. Franke T.F. Reed J.C. Science. 1999; 284: 339-343Crossref PubMed Scopus (962) Google Scholar). A catalytically inactive mutant of CnA that functions as a dominant negative mutant was prepared from ΔCnA by mutating histidine at position 160 to glutamine (19Wang H.-G. Pathan N. Ethell I.M. Krajewski S. Yamaguchi Y. Shibasaki F. Mckeon F. Bobo T. Franke T.F. Reed J.C. Science. 1999; 284: 339-343Crossref PubMed Scopus (962) Google Scholar). Hemagglutinin-tagged wild-type and mutant CnAβs were subcloned into an adenoviral vector downstream of the Tet-Off system (18Shibasaki F. MacKeon F. J. Cell Biol. 1995; 131: 735-743Crossref PubMed Scopus (229) Google Scholar). Cardiomyocytes were infected with adenoviral vectors at a multiplicity of infection of 100 plaque-forming units per cell in serum-free M199 medium for 2 h at 37 °C. Efficiency of infection under these conditions was 100%, as revealed by immunostaining with an anti-hemagglutinin antibody. In control cells, the expression of adenoviral vectors was suppressed with 1 μm doxycycline (DOX) added to culture medium. Yeast Two-hybrid Screen—We used Matchmaker 3 (Clontech) for the yeast two-hybrid screen. DNA fragments corresponding to the following segments of the central cytoplasmic loop of dog NCX1, shown in Fig. 1a, were prepared using a PCR-based method: the NCX inhibitory peptide (XIP, Fig. 1a), aa 250–406; β1, aa 407–478; β1-β2, aa 479–538; β2, aa 539–613; C terminus (CT, Fig. 1a), aa 614–796; N terminus (NT, Fig. 1a), aa 250–613; and the full loop (Full, Fig. 1a), aa 250–796. Each DNA fragment was fused to the GAL4 DNA binding domain in the pGBKT7 vector. We used a mixture of yeasts carrying each of these bait constructs for the initial screening of ∼4 × 107 clones from a human brain cDNA library fused to the GAL4 activation domain. These bait and library yeast strains were mated by culturing on agar plates in complete YPD (1% yeast extract, 2% peptone, and 2% glucose) medium overnight at 20 °C. After successive screening in medium lacking histidine, leucine, and tryptophan (-HLT, Fig. 1b) and then in medium lacking histidine, adenine, leucine, and tryptophan (-HALT, Fig. 1b), grown-up colonies were isolated and subjected to β-galactosidase assay according to the manufacturer's instructions. The inserts in β-galactosidase-positive clones were sequenced using the activation domain sequence primers by the ABI 9600 sequencer. Positive clones were verified by one-on-one transformations and selections by growth on agar plates in -HALT medium and β-galactosidase assay (see Fig. 1b). Immunoprecipitation, Immunoblot, and Immunocytochemical Analyses—Brain and/or ventricular tissues from rat or hamster and cultured rat cardiomyocytes were homogenized by Hiscotron (NITI-ON, Funabashi, Japan) in radioimmune precipitation assay lysis buffer containing 20 mm HEPES (pH 7.4), 150 mm NaCl, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 2 μg/ml leupeptin, 1 μg/ml aprotinin, 200 μm phenylmethylsulfonyl fluoride, and 200 μm benzamidine hydrochloride. The lysates were subjected to centrifugation at 100,000 × g for 20 min, and the resultant supernatant (up to 5 mg protein) was pre-cleared with 50 μl of protein A-Sepharose beads for 2 h at 4 °C on a rotator. After centrifugation, the supernatant was incubated with anti-pan CnA for 2 h at 4 °C and then with 50 μl of protein A-Sepharose beads for at least 2 h at 4 °C on a rotator. The beads were washed eight times with ice-cold phosphate-buffered saline. Proteins solubilized from beads by boiling in the Laemmli buffer (20Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206989) Google Scholar) were subjected to SDS-PAGE on a 8.5% gel and then to immunoblotting with an appropriate antibody. Immunoblot analysis was performed essentially as described previously (21Tawada-Iwata Y. Imagawa T. Yoshida A. Takahashi M. Nakamura H. Shigekawa M. Am. J. Physiol. 1993; 264: H1447-H1453PubMed Google Scholar). The immunoblot was visualized using an enhanced chemiluminescence detection system (Amersham Biosciences). For immunocytochemistry, 5-μm-thick sections of normal and BIO14.6 hamster ventricular tissues embedded in OCT compound (Tissue-Tek) were permeabilized with 0.1% Triton X-100 and treated with rabbit polyclonal anti-NCX1 or goat polyclonal anti-CnAβ at dilutions of 1:500 and 1:200, respectively. These samples were then treated with fluorescein isothiocyanate-conjugated anti-rabbit IgG or rhodamine-conjugated anti-goat IgG. For immunostaining of rat cardiomyocytes, cells immobilized on collagen-coated glass slides were fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.1% Triton X-100, and then stained with anti-NCX1, anti-pan CnA, or anti-ANP. For double staining with a combination of polyclonal (rabbit) and monoclonal (mouse) antibodies, fixed and permeabilized myocytes were incubated with a mixture of two primary antibodies and then with a mixture of the fluorescence-labeled anti-mouse and rhodamine-labeled anti-rabbits IgGs. Cells were examined by a confocal laser scanning microscopy (MRC-1024, Bio-Rad) mounted on an Olympus BX50WI epifluorescence microscope with a plan-apochromat 60× water immersion objective lens (Olympus). Fractionation of Heart Extracts—Normal and BIO14.6 hamster hearts were homogenized in phosphate-buffered saline using a Hiscotron homogenizer and centrifuged at 15,000 × g for 15 min. The resultant supernatant was centrifuged at 500,000 × g for 45 min to yield supernatant and pellet fractions. Most of the sarcolemmal and sarcoplasmic reticulum membranes were presumably recovered in the pellet fraction. Both the supernatant and pellet fractions were then subjected to immunoblot analysis with anti-pan CnA. Na+i-dependent 45Ca2+Uptake into and Na+o-dependent 45Ca2+Efflux from Cells—Na+i-dependent 45Ca2+ uptake into cells was measured as described previously (8Iwamoto T. Pan Y. Wakabayashi S. Imagawa T. Yamanaka H.I. Shigekawa M. J. Biol. Chem. 1996; 271: 13609-13615Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 9Iwamoto T. Pan Y. Nakamura T.Y. Wakabayashi S. Shigekawa M. Biochemistry. 1998; 37: 17230-17238Crossref PubMed Scopus (94) Google Scholar, 22Pan Y. Iwamoto T. Uehara A. Nakamura T.Y. Imanaga I. Shigekawa M. Am. J. Physiol. 2000; 279: C393-C402Crossref PubMed Google Scholar) with slight modifications. Cardiomyocytes or CCL39 cells cultured in 24-well dishes were loaded with Na+ by incubating them at 37 °C for 30 min in 0.5 ml of normal BSS (10 mm Hepes/Tris (pH7.4), 146 mm NaCl, 4 mm KCl, 2 mm MgCl2, 0.1 mm CaCl2, 10 mm glucose, and 0.1% bovine serum albumin) containing 1 mm ouabain and 10 μm monensin. In cardiomyocytes pretreated with PE or FCS, Na+ loading was carried out during the last 30 min of such pretreatment. 45Ca2+ uptake was then initiated by switching the medium to Na+-free BSS containing choline chloride or to normal BSS, both of which contained 370 kBq of 45Ca2+ and 1 mm ouabain. After a 30-s incubation, cells were washed with an ice-cold solution containing 10 mm LaCl3 to stop 45Ca2+ uptake. Cells were subsequently solubilized with 0.1 n NaOH, and aliquots were taken for the determination of radioactivity and protein. Na+i-dependent 45Ca2+ uptake was estimated by subtracting 45Ca2+ uptake in normal BSS from that in Na+-free BSS. To observe the effects of FK506, protein kinase modulators, or thapsigargin, cells were incubated with these substances during the last 15–30 min of Na+ loading, except that endogenous PKC was down-regulated by treatment with 0.3 μm PMA for 24 h. The Na+i-dependent 45Ca2+ uptake activities in control cells not pretreated with PE or other agents were as follows: for cardiomyocytes, 12.1 ± 0.5 nmol/mg/30s (n = 9); for CCL39 cells expressing the wild-type NCX1, NCX1Δ246–672, and NCX1Δ407–478, 10.4 ± 0.5, 4.2 ± 0.4, and 5.3 ± 0.2 nmol/mg/30s (n = 9), respectively. These values were taken as 100% in Figs. 3d, 4a, 5, 6, and 7.Fig. 5Effect of activated or dominant negative CnA on Na+i-dependent 45Ca2+ uptake in cardiomyocytes pretreated with PE or FCS. Myocytes were treated with no growth factor (-PE), 10 μm PE (+PE), or 10% FCS (+FCS) for 72 h. In one series (bar on the extreme right for +PE), PE-treated myocytes were subsequently infected with dominant negative can, and the rate of Na+i-dependent 45Ca2+ uptake was measured 24 h later. In all other series, myocytes were infected at 24 h after the start of growth factor treatment either with activated CnA in presence (+DOX/AdCnA) or absence (+AdCnA) of DOX or with dominant negative CnA (+AdD.N.CnA), and uptake rates were measured 48 h later. The uptake rate in the -PE+DOX/AdCnA series was taken as 100%. Data are averages ± S.D. (n = 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6Effects of FK506, protein kinase modulators and their combinations on Na+i-dependent 45Ca2+ uptake into PE-treated cardiomyocytes. Myocytes were incubated with the indicated agents during the last 30 min of a 72-h treatment with 0 or 10 μm PE, and then the rates of Na+i-dependent 45Ca2+ uptake were measured. In some series, myocytes were incubated with 0.3 μm PMA during the last 24 h of PE treatment (PMA(24h)). Cal.C, calphostin C. Data are averages ± S.D. (n = 3).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 7Effect of FK506 or PMA on Na+i-dependent 45Ca2+ uptake into CCL39 cells expressing NCX1 variants and/or activated CnA.a, cells expressing wild-type NCX1 or NCX1Δ246–672 were infected with activated CnA (AdCnA) for 48 h in the presence or absence of 1 μm DOX. The cells were then incubated with the indicated concentrations of FK506 or PMA for 30 min, and the rates of Na+i-dependent 45Ca2+ uptake were measured. The uptake rate in the series with activated CnA in the presence of DOX (+DOX/AdCnA) was taken as 100%. b, cells expressing wild-type NCX1, NCX1Δ407–478, or NCX1Δ246–672 were incubated with 1 μm thapsigargin (TG) and the indicated concentrations of FK506 for 30 min, and the uptake rates were measured. c, the uptake rate was measured as for panel b, except that thapsigargin was omitted. In panels b and c the uptake rate with no FK506 was taken as 100% for each NCX variant. Data are averages ± S.D. (n = 9). *, p < 0.05 versus control; **, p < 0.01 versus control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To measure 45Ca2+ efflux, cardiomyocytes in 35-mm dishes were incubated in 1 ml BSS containing 740 kBq of 45Ca2+ for the last 2 and 4 h of the 72-h treatment with and without 10 μm PE, respectively, which produced essentially the same level of 45Ca2+ loading in PE-treated and non-treated cells. After rinsing cells six times with Ca2+- and Na+-free BSS for 1 min, 45Ca2+ efflux was measured for 20 s in Ca2+- and Na+-free BSS or in Ca2+-free BSS, both of which contained 1 μm thapsigargin, to acutely increase [Ca2+]i (23Furukawa K. Tawada Y. Shigekawa M. J. Biol. Chem. 1988; 263: 8058-8065Abstract Full Text PDF PubMed Google Scholar). Na+o-dependent 45Ca2+ efflux was estimated by subtracting 45Ca2+ efflux in Ca2+- and Na+-free BSS from that in Ca2+-free BSS. Detection of TUNEL-positive Cells—For in situ detection of DNA fragmentation, we assayed TUNEL-positive cells using an apoptosis detection kit (Takara Biomedical) essentially as described previously (24Saito S. Hiroi Y. Zou Y. Aikawa R. Toko H. Shibasaki F. Yazaki Y. Nagai R. Komuro I. J. Biol. Chem. 2000; 275: 34528-34533Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). The number of TUNEL-labeled nuclei was counted by observing rat cardiomyocytes with a light microscope (40× objective; Olympus). TUNEL-positive cells were <3% in normal cardiomyocytes. Data Analysis—Reproducibility of the data presented in the figures and those described in the text was confirmed in at least three independent experiments. Significant differences between two groups of data were evaluated by an analysis of variance assay with post hoc tests. p < 0.05 was considered as a statistically significant finding. Isolation of CnAβ as a NCX1-binding Protein and Mapping of the Interacting Site in NCX1—To isolate protein(s) interacting with NCX1, we performed the yeast two-hybrid screen of a human brain cDNA library using various segments of the large central loop of NCX1 as bait (Fig. 1a). From an initial screen in which we used a mixture of yeast populations expressing individual bait sequences, we isolated a positive clone encoding a ∼100 amino acid C-terminal tail of CnAβ with its autoinhibitory domain (Fig. 1a). We then examined the interaction of individual NCX segments with the CnAβ tail by one-on-one transformations and selection by colony growth and β-galactosidase assays (Fig. 1b). We confirmed that aa 407–478 of the NCX1 protein, known as the β1 repeat, and other fragments containing this same sequence associate with the CnAβ tail. We examined whether calcineurin interacts with NCX1 and its isoforms (NCX2 and NCX3) at the protein level. We found that anti-pan CnA co-precipitated proteins reactive with antibody to each isoform from lysates of rat brain and heart, although these proteins were still relatively abundant in the supernatant fractions (Fig. 1c). Thus, at least some calcineurin was physically associated with NCX isoforms, consistent with the fact that the β1 repeat sequence is conserved in these isoforms. Of note, antipan CnA immunoprecipitated single major proteins from rat (Fig. 1c) and hamster (Fig. 2b) hearts that were recognized by anti-CnAβ, indicating that the antibody predominantly precipitated CnAβ under the conditions used. Enhanced Association of CnAβ with NCX1 in BIO14.6 Hamster Heart—The BIO14.6 hamsters develop cardiomyopathy and muscular dystrophy due to δ-sarcoglycan deficiency (25Bajusz E. Homburger F. Baker J.R. Bogdonoff P. Ann. N. Y. Acad. Sci. 1969; 156: 396-420Crossref PubMed Scopus (29) Google Scholar, 26Nigro V. Okazaki Y. Belsito A. Piluso G. Matsuda Y. Politano L. Nigro G. Ventura C. Abbondanza C. Molinari A.M. Acampora D. Nishimura M. Hayashizaki Y. Puca G.A. Hum. Mol. Genet. 1997; 6: 601-607Crossref PubMed Scopus (247) Google Scholar). We examined the interaction of NCX1 with calcineurin in the hearts of 120-day-old BIO14.6 hamsters, because our recent study has suggested that [Ca2+]i might be elevated in BIO14.6 cardiomyocytes due to increased basal Ca2+ influx (27Iwata Y. Katanosaka Y. Arai Y. Komamura K. Miyatake K. Shigekawa M. J. Cell Biol. 2003; 161: 957-967Crossref PubMed Scopus (218) Google Scholar). Anti-CnAβ immunoprecipitated more abundant NCX1 protein from BIO14.6 than from normal hearts despite the similar contents of NCX1 and calcineurin in these preparations (Fig. 2b). Interestingly, calmodulin also was more abundant in the immunoprecipitates from BIO14.6 heart, although total calmodulin was again not different (Fig. 2b), suggesting that calcineurin is activated in the BIO14.6 heart. Immunocytochemistry with anti-CnAβ revealed the presence of calcineurin at the peripheral sarcolemma in BIO14.6 but not in normal cardiomyocytes, although it was detectable in the cell interior and at the intercalated discs in both types of myocytes (Fig. 2a). Striated patterns seen in the cell interior may reflect the presence of calcineurin in the Z-lines (16Vega R.B. Bassel-Duby R. Olson E.N. J. Biol. Chem. 2003; 278: 36981-36984Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Furthermore, calcineurin was more abundant in the membrane versus the cytosolic fraction prepared from the BIO14.6 h" @default.
• W2000398093 created "2016-06-24" @default.
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• W2000398093 title "Calcineurin Inhibits Na+/Ca2+ Exchange in Phenylephrine-treated Hypertrophic Cardiomyocytes" @default.
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