Calcineurin Inhibits Na+/Ca2+ Exchange in Phenylephrine-treated Hypertrophic Cardiomyocytes

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. 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 (230) Google Scholar, 2Bers D.M. Circ. Res. 2000; 87: 275-281Crossref PubMed Scopus (479) 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 (212) 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 (521) 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 (176) Google Scholar, 9Iwamoto T. Pan Y. Nakamura T.Y. Wakabayashi S. Shigekawa M. Biochemistry. 1998; 37: 17230-17238Crossref PubMed Scopus (95) 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 (95) 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 (95) 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 (2241) Google Scholar, 15Molkentin J.D. Dorn II, G.W. Ann. Rev. Physiol. 2001; 63: 391-426Crossref PubMed Scopus (581) Google Scholar, 16Vega R. Olson E.N. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), NCX might contribute to the etiology of in vivo cardiac PE, PMA, Rp-8-CPT-cAMPS, Rp-8-CPT-cGMPS, and were from and were from was from to NCX have been (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 (176) Google Scholar, 9Iwamoto T. Pan Y. Nakamura T.Y. Wakabayashi S. Shigekawa M. Biochemistry. 1998; 37: 17230-17238Crossref PubMed Scopus (95) Google Scholar). calcineurin A and natriuretic and and and were the from the in was from was from and and normal were from The the as the for the of a for were from of as A. S. Circ. Res. PubMed Scopus Google Scholar). were on a of cells well and in with with that of the cells were cardiomyocytes. the cells were and for to in with PE, or with (see Fig. and beating A. S. Circ. Res. PubMed Scopus Google Scholar). On the other hand, CCL39 cells and their NCX1 were in containing FCS, and of and of NCX1 and The of of heart NCX1 and NCX1 mutants or their CCL39 and the of expressing NCX activity were as (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 (176) Google Scholar, 9Iwamoto T. Pan Y. Nakamura T.Y. Wakabayashi S. Shigekawa M. Biochemistry. 1998; 37: 17230-17238Crossref PubMed Scopus (95) Google Scholar). The of human and the of its the autoinhibitory and the were J. Biol. PubMed Scopus (230) Google Scholar, N. S. Y. T. Science. 1999; PubMed Scopus Google Scholar). A of that functions as a was from by to N. S. Y. T. Science. 1999; PubMed Scopus Google Scholar). and were an of the J. Biol. PubMed Scopus (230) Google Scholar). were with a of of in for of under these conditions was as by with an In the expression of was with to used for the yeast two-hybrid screen. to the of the central cytoplasmic loop of NCX1, shown in Fig. were using a the NCX Fig. C terminus Fig. terminus Fig. and the loop Fig. was to the in the used a of of these for the of from a human to the activation and yeast were by on in yeast and in and Fig. and in and Fig. were isolated and subjected to to the The in were using the activation by the were by and by growth on in and (see Fig. and from rat or hamster and rat cardiomyocytes were by in containing and The were subjected to for and the to was with of protein for on a the was with for and with of protein for on a The were with from by in the PubMed Scopus Google were subjected to on a and to with an was as Y. Imagawa T. A. M. Nakamura H. Shigekawa M. Am. J. Physiol. 1993; Google Scholar). The was using an enhanced For of normal and BIO14.6 hamster in were with and with or of and were with or For of rat cells on were with for with and with CnA, or For with a of and and were with a of primary and with a of the and were by a on an with a of and BIO14.6 hamster hearts were in using a and for The was for to and of the sarcolemmal and sarcoplasmic reticulum were presumably in the the and were subjected to with Na+i-dependent and Na+o-dependent from 45Ca2+ uptake cells was measured as (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 (176) Google Scholar, 9Iwamoto T. Pan Y. Nakamura T.Y. Wakabayashi S. Shigekawa M. Biochemistry. 1998; 37: 17230-17238Crossref PubMed Scopus (95) Google Scholar, Y. Iwamoto T. A. Nakamura T.Y. Shigekawa M. Am. J. Physiol. 2000; PubMed Google with or CCL39 cells in were with Na+ by for in of normal and containing and In cardiomyocytes with PE or FCS, Na+ was during the of such pretreatment. 45Ca2+ uptake was by the to containing or to normal BSS, both of which of 45Ca2+ and a cells were with an containing to 45Ca2+ were with and were for the of and Na+i-dependent 45Ca2+ uptake was by 45Ca2+ uptake in normal from that in the effects of protein kinase or cells were with these during the of Na+ that endogenous PKC was by treatment with PMA for The Na+i-dependent 45Ca2+ uptake activities in cells not with PE or other were as for for CCL39 cells expressing the NCX1, and and were as in and of or on Na+i-dependent 45Ca2+ uptake in cardiomyocytes with PE or were with growth PE or for In on the for were with and the rate of Na+i-dependent 45Ca2+ uptake was measured In other were the of growth treatment with in or of or with and uptake were measured The uptake rate in the was as are of protein kinase and their on Na+i-dependent 45Ca2+ uptake cardiomyocytes. were with the during the of a treatment with or PE, and the of Na+i-dependent 45Ca2+ uptake were In some were with PMA during the of PE treatment are of or PMA on Na+i-dependent 45Ca2+ uptake CCL39 cells expressing NCX1 cells expressing NCX1 or were with for in the or of The cells were with the of or PMA for and the of Na+i-dependent 45Ca2+ uptake were The uptake rate in the with in the of was as cells expressing NCX1, or were with and the of for and the uptake were the uptake rate was measured as for that was In and the uptake rate with was as for NCX are 45Ca2+ cardiomyocytes in were in containing of 45Ca2+ for the and of the treatment with and PE, which the of 45Ca2+ in and cells with and for 45Ca2+ was measured for in and or in BSS, both of which to Ca2+i K. 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Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). calcineurin was in the membrane the from the BIO14.6 the was of normal heart the association of calcineurin with NCX1 is significantly enhanced in BIO14.6 compared with normal NCX by in or CCL39 the effects of calcineurin on NCX we used rat neonatal cardiomyocytes subjected to with PE for (see in Fig. In these a in with enhanced and enhanced expression of was observed (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 (2241) Google Scholar, A. S. Circ. Res. PubMed Scopus Google Scholar, T. J.D. S. A. 2000; PubMed Scopus Google Scholar). The NCX1 protein was in the in the and and its expression increased during the PE treatment, although calcineurin expression observed of NCX1 and calcineurin in the of a PE treatment, consistent with the that a of NCX1 protein was in the from the did not a of the of cells was the in the rate of Na+i-dependent 45Ca2+ uptake measured as activity of protein was markedly in a PE treatment The uptake rate in the be was to NCX1 (see On the other hand, the uptake rate was increased in a PE This uptake may be to the increased NCX1 expression this were with the calcineurin during the of the PE treatment, the rate of Na+i-dependent 45Ca2+ uptake increased were with with the rate of Na+i-dependent 45Ca2+ uptake also increased by A, calcineurin inhibitor, was to the On the other hand, also enhanced the rate of Na+o-dependent Ca2+ from cardiomyocytes In this we and with 45Ca2+ to of and 45Ca2+ was by Ca2+i with under conditions. data that PE and both the and of NCX activity. the of calcineurin in NCX we the effect of the of or on cardiomyocytes with PE or for to (see Fig. CnA, the of PE treatment, a large in the rate of Na+i-dependent 45Ca2+ uptake an in the of the hypertrophic On the other hand, the of the PE or treatment, the of the and uptake inhibition and depressed myocyte effects on not with growth factors and data not significantly increased the of as reported T. J.D. S. A. 2000; PubMed Scopus Google Scholar, S. T. J.D. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google and reduced the uptake rate in these cells by the uptake although little effect on a of NCX inhibition occurring in or is to the activity of the effects of protein kinase on the rate of Na+i-dependent Ca2+ uptake in chronically of with PMA during the of a PE treatment little effect on the uptake rate a in not consistent with a (9Iwamoto T. Pan Y. Nakamura T.Y. Wakabayashi S. Shigekawa M. Biochemistry. 1998; 37: 17230-17238Crossref PubMed Scopus (95) Google Scholar). In with PMA during the of a PE treatment an in the uptake rate to that The PMA effect was by a with PKC the uptake rate in the of C or increased by and as compared with that of The effects of and PMA treatment or C were that the activities of calcineurin and PKC independently to the observed of uptake activity in the protein kinase A the protein kinase A and the protein kinase the protein kinase and the protein kinase and did not the uptake rate and data not using CCL39 cells expressing NCX1 we the role of the central cytoplasmic loop of the exchanger in the of NCX inhibition by the of calcineurin and In cells expressing NCX1 with for the rate of Na+i-dependent 45Ca2+ uptake was compared with that for cells in which expression of been with such uptake was reversed by significantly by uptake by calcineurin and PKC were in CCL39 cells as in cardiomyocytes. However, in cells expressing an NCX1 of its central loop the uptake rate was not by or PMA that the central loop is required for the effects of calcineurin and used CCL39 cells not with to the interaction of endogenous calcineurin with the β1 repeat deletion In was to cells the uptake to a low Ca2+i by the of the reticulum endogenous calcineurin was not in The rate of Na+i-dependent 45Ca2+ uptake in cells expressing NCX1 was and with in and compared with in the of In little effect on cells expressing or of treatment and effects of on NCX activity were that the β1 repeat is required for the action of endogenous of NCX by and have that of the and are expressed in with the a role in the of cardiac hypertrophy T. J.D. S. A. 2000; PubMed Scopus Google Scholar, J.D. S. A. PubMed Scopus Google Scholar). we have that the of containing an autoinhibitory binds to the β1 repeat of NCX1, of repeat in the central cytoplasmic loop of NCX S. S. A. PubMed Scopus Google Scholar). The β1 repeat constitutes part of the Ca2+ regulatory that is for the allosteric regulation of NCX activity by intracellular Ca2+ (see M. Iwamoto T. Circ. Res. 2001; 88: 864-876Crossref PubMed Scopus (212) Google Scholar). binds to a important of the the association of the proteins was enhanced in BIO14.6 cardiomyopathic hamster heart enhanced association of calcineurin with was also that calcineurin is to a in Ca2+i in BIO14.6 cardiomyocytes. an is consistent with our Y. Y. Y. K. K. Shigekawa M. J. Biol. 2003; PubMed Scopus Google Scholar). used neonatal rat cardiomyocytes with PE or for as an in hypertrophic (see Fig. The hypertrophic by increased enhanced and increased although treatment S. and M. the NCX activity in these hypertrophic which was measured as the rate of Na+i-dependent 45Ca2+ uptake or the rate of Na+o-dependent Ca2+ was markedly to in and depressed activity was partially reversed by A, or with and On the other hand, a in NCX activity in or although little effect on cells the of both NCX inhibition and hypertrophy in or and we suggest that calcineurin activity is elevated in hypertrophic as reported T. J.D. S. A. 2000; PubMed Scopus Google and that this activity causes NCX PKC also a of depressed NCX activity in and The effects of the of calcineurin and PKC were that the of these are and C with little on myocyte hypertrophic phenotypes. is that these enzyme via involving the NCX inhibition requires activation of with the NCX inhibition and the NCX inhibition the of CCL39 cells with S. and M. S. T. J.D. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J.D. J. Biol. PubMed Scopus Google have that hypertrophic calcineurin is with of and and that is a mediator of the hypertrophy of isolated rat and cardiomyocytes. The NCX inhibition by calcineurin and PKC in cardiomyocytes was in CCL39 cells expressing cloned NCX1, not in expressing that the central cytoplasmic loop of NCX1 is required for the effects of calcineurin and this the view that the observed NCX inhibition from changes in cellular such as across the plasma membrane. that PE the of the exchanger NCX This effect of PE, with the acute NCX stimulation by PE or other agonists of Gαq-coupled receptors reported (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 (176) Google Scholar, 9Iwamoto T. Pan Y. Nakamura T.Y. Wakabayashi S. Shigekawa M. Biochemistry. 1998; 37: 17230-17238Crossref PubMed Scopus (95) Google Scholar, 10Stengl M. Mubagwa K. Carmeliet E. Flameng W. Cardiovasc. Res. 1998; 38: 703-710Crossref PubMed Scopus (40) Google Scholar), that regulatory for cardiac observed that the β1 repeat deletion from NCX1 expressed in CCL39 cells the effect of endogenous the deletion of the large cytoplasmic loop the of both endogenous calcineurin and data are consistent with the view that NCX1 is for the effect of endogenous that the large cytoplasmic loop of NCX1 may be required to maintain the interaction of calcineurin with its The in the cytoplasmic loop might serve as the calcineurin the cytoplasmic loop could function as a for ancillary protein(s) that might serve as the substrate. might be for the endogenous calcineurin to the β1 repeat as well as for the in although this may not the large cytoplasmic loop is is that CnA, its and the ability to to the NCX1 β1 was to NCX activity in view of the in these we the that calcineurin may NCX activity to are required to the calcineurin and NCX of Depressed NCX hypertrophy occurs in to a variety of including the chronic activation of of that leads to the activation of PKC, and protein J.D. Dorn II, G.W. Ann. Rev. Physiol. 2001; 63: 391-426Crossref PubMed Scopus (581) Google Scholar). On the other hand, calcineurin and its primary the of cells have been shown to be important of in and in vivo cardiac hypertrophic (see 15Molkentin J.D. Dorn II, G.W. Ann. Rev. Physiol. 2001; 63: 391-426Crossref PubMed Scopus (581) Google and 16Vega R. Olson E.N. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The mechanism by which calcineurin pathological hypertrophic is as for calcineurin other the that contribute to the of cardiac hypertrophy have been This has that NCX1 may be of such for The shown are in with the by Z. B. W. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google that NCX was significantly reduced in hypertrophic cardiomyocytes isolated from this did not in A during Because depressed NCX activity is to the chronic of Ca2+i in depressed NCX activity might an important role in the of hypertrophy and the subsequent However, the pathological of depressed NCX activity to be in as enhanced NCX1 expression and function have also been reported for 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).