title: Molecular basis of calpain cleavage and inactivation of the sodium-calcium exchanger 1 in heart failure.
abstract: Cardiac sodium (Na(+))-calcium (Ca(2+)) exchanger 1 (NCX1) is central to the maintenance of normal Ca(2+) homeostasis and contraction. Studies indicate that the Ca(2+)-activated protease calpain cleaves NCX1. We hypothesized that calpain is an important regulator of NCX1 in response to pressure overload and aimed to identify molecular mechanisms and functional consequences of calpain binding and cleavage of NCX1 in the heart. NCX1 full-length protein and a 75-kDa NCX1 fragment along with calpain were up-regulated in aortic stenosis patients and rats with heart failure. Patients with coronary artery disease and sham-operated rats were used as controls. Calpain co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes and left ventricle lysate. Immunoprecipitations, pull-down experiments, and extensive use of peptide arrays indicated that calpain domain III anchored to the first Ca(2+) binding domain in NCX1, whereas the calpain catalytic region bound to the catenin-like domain in NCX1. The use of bioinformatics, mutational analyses, a substrate competitor peptide, and a specific NCX1-Met(369) antibody identified a novel calpain cleavage site at Met(369). Engineering NCX1-Met(369) into a tobacco etch virus protease cleavage site revealed that specific cleavage at Met(369) inhibited NCX1 activity (both forward and reverse mode). Finally, a short peptide fragment containing the NCX1-Met(369) cleavage site was modeled into the narrow active cleft of human calpain. Inhibition of NCX1 activity, such as we have observed here following calpain-induced NCX1 cleavage, might be beneficial in pathophysiological conditions where increased NCX1 activity contributes to cardiac dysfunction.
authors.affiliation: From the Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, 0407 Oslo, Norway, the KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, 0318 Oslo, Norway.
authors.forename: Pimthanya
authors.initials: P
authors.lastname: Wanichawan
authors.affiliation: From the Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, 0407 Oslo, Norway, the KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, 0318 Oslo, Norway.
authors.forename: Tandekile Lubelwana
authors.initials: TL
authors.lastname: Hafver
authors.affiliation: From the Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, 0407 Oslo, Norway, the KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, 0318 Oslo, Norway.
authors.forename: Kjetil
authors.initials: K
authors.lastname: Hodne
authors.affiliation: From the Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, 0407 Oslo, Norway, Bjorknes College, 0456 Oslo, Norway.
authors.forename: Jan Magnus
authors.initials: JM
authors.lastname: Aronsen
authors.affiliation: From the Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, 0407 Oslo, Norway, the KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, 0318 Oslo, Norway, the Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Ida Gjervold
authors.initials: IG
authors.lastname: Lunde
authors.affiliation: the Departments of Microbiology and Medical Biochemistry, Oslo University Hospital, Rikshospitalet, 0372 Oslo, Norway, and.
authors.forename: Bjørn
authors.initials: B
authors.lastname: Dalhus
authors.affiliation: From the Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, 0407 Oslo, Norway, the KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, 0318 Oslo, Norway.
authors.forename: Marianne
authors.initials: M
authors.lastname: Lunde
authors.affiliation: From the Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, 0407 Oslo, Norway, the KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, 0318 Oslo, Norway.
authors.forename: Heidi
authors.initials: H
authors.lastname: Kvaløy
authors.affiliation: From the Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, 0407 Oslo, Norway, the KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, 0318 Oslo, Norway.
authors.forename: William Edward
authors.initials: WE
authors.lastname: Louch
authors.affiliation: From the Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, 0407 Oslo, Norway, the Department of Cardiothoracic Surgery, Oslo University Hospital, Ullevål, 0407 Oslo, Norway.
authors.forename: Theis
authors.initials: T
authors.lastname: Tønnessen
authors.affiliation: From the Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, 0407 Oslo, Norway, the KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, 0318 Oslo, Norway.
authors.forename: Ivar
authors.initials: I
authors.lastname: Sjaastad
authors.affiliation: From the Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, 0407 Oslo, Norway, the KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, 0318 Oslo, Norway.
authors.forename: Ole Mathias
authors.initials: OM
authors.lastname: Sejersted
authors.affiliation: From the Institute for Experimental Medical Research, Oslo University Hospital and University of Oslo, 0407 Oslo, Norway, the KG Jebsen Cardiac Research Center and Center for Heart Failure Research, University of Oslo, 0318 Oslo, Norway, c.r.carlson@medisin.uio.no.
authors.forename: Cathrine Rein
authors.initials: CR
authors.lastname: Carlson
mesh_terms: D000368:Aged; D000595:Amino Acid Sequence; D000818:Animals; D001024:Aortic Valve Stenosis / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D001665:Binding Sites; D002154:Calpain / Q000235:genetics / Q000378:metabolism*; D005260:Female; D057809:HEK293 Cells; D006333:Heart Failure / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D006352:Heart Ventricles / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism / Q000473:pathology; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000473:pathology; D061251:Primary Cell Culture; D011485:Protein Binding; D059748:Proteolysis; D051381:Rats; D017208:Rats, Wistar; D019831:Sodium-Calcium Exchanger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; D002154:Calpain; C484806:CAPN1 protein, human
keywords: Animal Model; Aortic Stenosis; Calpain; Computer Modeling; Electrophysiology; Heart Failure; Ion Channel; Peptide Array; Protein-Protein Interaction; Sodium-Calcium Exchanger