title: Different kinetics of the regulation of respiration in permeabilized cardiomyocytes and in HL-1 cardiac cells. Importance of cell structure/organization for respiration regulation.
abstract: The aim of this study was to investigate the mechanism of cellular regulation of mitochondrial respiration in permeabilized cardiac cells with clearly different structural organization: (i) in isolated rat cardiomyocytes with very regular mitochondrial arrangement, (ii) in HL-1 cells from mouse heart, and (iii) in non-beating (NB HL-1 cells) without sarcomeres with irregular and dynamic filamentous mitochondrial network. We found striking differences in the kinetics of respiration regulation by exogenous ADP between these cells: the apparent Km for exogenous ADP was by more than order of magnitude (14 times) lower in the permeabilized non-beating NB HL-1 cells without sarcomeres (25+/-4 microM) and seven times lower in normally cultured HL-1 cells (47+/-15 microM) than in permeabilized primary cardiomyocytes (360+/-51 microM). In the latter cells, treatment with trypsin resulted in dramatic changes in intracellular structure that were associated with 3-fold decrease in apparent Km for ADP in regulation of respiration. In contrast to permeabilized cardiomyocytes, in NB HL-1 cells creatine kinase activity was low and the endogenous ADP fluxes from MgATPases recorded spectrophotometrically by the coupled enzyme assay were not reduced after activation of mitochondrial oxidative phosphorylation by the addition of mitochondrial substrates, showing the absence of ADP channelling in the NB HL-1 cells. While in the permeabilized cardiomyocytes creatine strongly activated mitochondrial respiration even in the presence of powerful competing pyruvate kinase-phosphoenolpyruvate system, in the NB HL-1 cells the stimulatory effect of creatine was not significant. The results of this study show that in normal adult cardiomyocytes and HL-1 cells intracellular local restrictions of diffusion of adenine nucleotides and metabolic feedback regulation of respiration via phosphotransfer networks are different, most probably related to differences in structural organization of these cells.
authors.affiliation: Laboratory of Bioenergetics, National Institute of Chemical Physics and Biophysics, Tallinn, Estonia.
authors.forename: Tiia
authors.initials: T
authors.lastname: Anmann
authors.forename: Rita
authors.initials: R
authors.lastname: Guzun
authors.forename: Nathalie
authors.initials: N
authors.lastname: Beraud
authors.forename: Sophie
authors.initials: S
authors.lastname: Pelloux
authors.forename: Andrey V
authors.initials: AV
authors.lastname: Kuznetsov
authors.forename: Lembi
authors.initials: L
authors.lastname: Kogerman
authors.forename: Tuuli
authors.initials: T
authors.lastname: Kaambre
authors.forename: Peeter
authors.initials: P
authors.lastname: Sikk
authors.forename: Kalju
authors.initials: K
authors.lastname: Paju
authors.forename: Nadja
authors.initials: N
authors.lastname: Peet
authors.forename: Enn
authors.initials: E
authors.lastname: Seppet
authors.forename: Carlos
authors.initials: C
authors.lastname: Ojeda
authors.forename: Yves
authors.initials: Y
authors.lastname: Tourneur
authors.forename: Valdur
authors.initials: V
authors.lastname: Saks
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002463:Cell Membrane Permeability; D019069:Cell Respiration; D003402:Creatine Kinase / Q000378:metabolism; D004735:Energy Transfer; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D051379:Mice; D018613:Microscopy, Confocal; D008929:Mitochondria, Heart / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
chemical_list: D000244:Adenosine Diphosphate; D003402:Creatine Kinase