Molecular and Cellular Biochemistry

, Volume 256, Issue 1–2, pp 13–27 | Cite as

Phosphotransfer dynamics in skeletal muscle from creatine kinase gene-deleted mice

  • Petras P. Dzeja
  • Andre Terzic
  • Be Wieringa


To assess the significance of energy supply routes in cellular energetic homeostasis, net phosphoryl fluxes catalyzed by creatine kinase (CK), adenylate kinase (AK) and glycolytic enzymes were quantified using 18O-phosphoryl labeling. Diaphragm muscle from double M-CK/ScCKmit knockout mice exhibited virtually no CK-catalyzed phosphotransfer. Deletion of the cytosolic M-CK reduced CK-catalyzed phosphotransfer by 20%, while the absence of the mitochondrial ScCKmit isoform did not affect creatine phosphate metabolic flux. Contribution of the AK-catalyzed phosphotransfer to total cellular ATP turnover was 15.0, 17.2, 20.2 and 28.0% in wild type, ScCKmit, M-CK and M-CK/ScCKmit deficient muscles, respectively. Glycolytic phosphotransfer, assessed by G-6-P 18O-phosphoryl labeling, was elevated by 32 and 65% in M-CK and M-CK/ScCKmit deficient muscles, respectively. Inhibition of glyceraldehyde 3-phosphate dehydrogenase (GAPDH)/phosphoglycerate kinase (PGK) in CK deficient muscles abolished inorganic phosphate compartmentation and redirected high-energy phosphoryl flux through the AK network. Under such conditions, AK phosphotransfer rate was equal to 86% of the total cellular ATP turnover concomitant with almost normal muscle performance. This indicates that near-equilibrium glycolytic phosphotransfer reactions catalyzed by the GAPDH/PGK support a significant portion of the high-energy phosphoryl transfer in CK deficient muscles. However, CK deficient muscles displayed aberrant AT-Pase-ATPsynthase communication along with lower energetic efficiency (P/O ratio), and were more sensitive to metabolic stress induced by chemical hypoxia. Thus, redistribution of phosphotransfer through glycolytic and AK networks contributes to energetic homeostasis in muscles under genetic and metabolic stress complementing loss of CK function.

energy metabolism metabolic networks adenylate kinase glycolysis gene knockout 18O- phosphoryl exchange 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Saks VA, Khuchua ZA, Vasilyeva EV, Belikova OY, Kuznetsov AV: Metabolic compartmentation and substrate channeling in muscle cells. Role of coupled creatine kinases in in vivo regulation of cellular respiration — a synthesis. Mol Cell Biochem 133–134: 155–192, 1994CrossRefGoogle Scholar
  2. 2.
    Wallimann T, Wyss M, Brdiczka D, Nicolay K, Eppenberger HM: Intracellular compartmentation, structure and function of creatine kinase isoenzymes in tissues with high and fluctuating energy demands: The ‘phosphocreatine circuit’ for cellular energy homeostasis. Biochem J 28: 21–40, 1992Google Scholar
  3. 3.
    Bessman SP, Geiger PJ: Compartmentation of hexokinase and creatine phosphokinase, cellular regulation, and insulin action. Curr Top Cell Reg 16: 55–86, 1980Google Scholar
  4. 4.
    van Deursen J, Heerschap A, Oerlemans F, Ruitenbeek W, Jap P, ter Laak H, Wieringa B: Skeletal muscle of mice deficient in muscle creatine kinase lack burst activity. Cell 74: 621–631, 1993CrossRefPubMedGoogle Scholar
  5. 5.
    Ingwall JS: Whole-organ enzymology of the creatine kinase system in heart. Biochem Soc Trans 19: 1006–1010, 1991PubMedGoogle Scholar
  6. 6.
    Dzeja PP, Zeleznikar RJ, Goldberg ND: Adenylate kinase: Kinetic behavior in intact cells indicates it is integral to multiple cellular processes. Mol Cell Biochem 84: 169–182, 1998CrossRefGoogle Scholar
  7. 7.
    Dzeja PP, Redfield MM, Burnett JC, Terzic A: Failing energetics in failing hearts. Curr Cardiol Rep 2: 212–217, 2000PubMedGoogle Scholar
  8. 8.
    Savabi F: Interaction of creatine kinase and adenylate kinase systems in muscle cells. Mol Cell Biochem 133–134: 145–152, 1994CrossRefGoogle Scholar
  9. 9.
    Laterveer FD, Nicolay K, Gellerich FN: Experimental evidence for dynamic compartmentation of ADP at the mitochondrial periphery: Coupling of mitochondrial adenylate kinase and mitochondrial hexokinase with oxidative phosphorylation under conditions mimicking the intracellular colloid osmotic pressure. Mol Cell Biochem 174: 43–51, 1997CrossRefPubMedGoogle Scholar
  10. 10.
    Dzeja PP, Pucar D, Redfield MM, Burnett JC, Terzic A: Reduced activity of enzymes coupling ATP-generating with ATP-consuming processes in the failing myocardium. Mol Cell Biochem 201: 33–40, 1999CrossRefPubMedGoogle Scholar
  11. 11.
    Kingsley-Hickman PB, Sako EY, Mohanakrishnan P, Robitaille PML, From AHL, Foker JE, Ugurbil K: 31P NMR studies of ATP synthesis and hydrolysis kinetics in the intact myocardium. Biochemistry 26: 7501–7510, 1987CrossRefPubMedGoogle Scholar
  12. 12.
    Joubert F, Mazet JL, Mateo P, Hoerter JA: 31P NMR detection of subcellular creatine kinase fluxes in the perfused rat heart: Contractility modifies energy transfer pathways. J Biol Chem 277: 18469–18476, 2002CrossRefPubMedGoogle Scholar
  13. 13.
    Van Dorsten FA, Nederhoff MG, Nicolay K, Van Echteld CJ: 31P NMR studies of creatine kinase flux in M-creatine kinase-deficient mouse heart. Am J Physiol 275: H1191–H1199, 1998PubMedGoogle Scholar
  14. 14.
    Dzeja PP, Zeleznikar RJ, Goldberg ND: Suppression of creatine kinase-catalyzed phosphotransfer results in increased phosphoryl transfer by adenylate kinase in intact skeletal muscle. J Biol Chem 271: 12847–12851, 1996CrossRefPubMedGoogle Scholar
  15. 15.
    Roman BB, Meyer RA, Wiseman RW: Phosphocreatine kinetics at the onset of contractions in skeletal muscle of MM creatine kinase knockout mice. Am J Physiol Cell Physiol 283: C1776–C1783, 2002PubMedGoogle Scholar
  16. 16.
    Mitchell P: Compartmentation and communication in living systems. Ligand conduction: A general catalytic principle in chemical, osmotic and chemiosmotic reaction systems. Eur J Biochem 95: 1–20, 1979CrossRefPubMedGoogle Scholar
  17. 17.
    Reich JG, Sel'kov EE: In: Energy Metabolism of the Cell: A Theoretical Treatise. Academic Press, London, 1981, pp 95–107Google Scholar
  18. 18.
    Dos Santos P, Aliev MK, Diolez P, Duclos F, Besse P, Bonoron-Adele S, Sikk P, Canioni P, Saks VA: Metabolic control of contractile performance in isolated perfused rat heart. Analysis of experimental data by reaction-diffusion mathematical model. J Mol Cell Cardiol 32: 1703–1734, 2000CrossRefPubMedGoogle Scholar
  19. 19.
    Dzeja PP, Vitkevicius KT, Redfield MM, Burnett JC, Terzic A: Adenylate kinase catalyzed phosphotransfer in the myocardium: Increased contribution in heart failure. Circ Res 84: 1137–1143, 1999PubMedGoogle Scholar
  20. 20.
    Dzeja PP, Kalvenas A, Toleikis A, Praskevicius A: The effect of adenylate kinase activity on the rate and efficiency of energy transport from mitochondria to hexokinase. Biochem Int 10: 259–265, 1985PubMedGoogle Scholar
  21. 21.
    Dzeja PP, Bortolon R, Perez-Terzic C, Holmuhamedov EL, Terzic A: Energetic communication between mitochondria and nucleus directed by catalyzed phosphotransfer. Proc Natl Acad Sci USA 99: 10156–10161, 2002CrossRefPubMedGoogle Scholar
  22. 22.
    Dzeja PP, Terzic A: Phosphotransfer reactions in the regulation of ATP-sensitive K+ channels. FASEB J 12: 523–529, 1998PubMedGoogle Scholar
  23. 23.
    Abraham MR, Selivanov VA, Hodgson DM, Pucar D, Zingman LV, Wieringa B, Dzeja PP, Alekseev AE, Terzic A: Coupling of cell energetics with membrane metabolic sensing. Integrative signaling through creatine kinase phosphotransfer disrupted by M-CK gene knock-out. J Biol Chem 277: 24427–24434, 2002CrossRefPubMedGoogle Scholar
  24. 24.
    Carrasco AJ, Dzeja PP, Alekseev AE, Pucar D, Zingman LV, Abraham MR, Hodgson D, Bienengraeber M, Puceat M, Janssen E, Wieringa B, Terzic A: Adenylate kinase phosphotransfer communicates cellular energetic signals to ATP-sensitive potassium channels. Proc Natl Acad Sci USA 98: 7623–7628, 2001CrossRefPubMedGoogle Scholar
  25. 25.
    Sasaki N, Sato T, Marban E, O'Rourke B: ATP consumption by uncoupled mitochondria activates sarcolemmal KATP channels in cardiac myocytes. Am J Physiol 280: H1882–H1888, 2001PubMedGoogle Scholar
  26. 26.
    Wallimann T, Schnyder T, Schlegel J, Wyss M, Wegman G, Rossi AM, Hemmer W, Eppenberger HM, Quest AFG: Subcellular compartmentation of creatine kinase isoenzymes, regulation of CK and octameric structure of mitochondrial CK: Important aspects of the phosphorylcreatine circuit. In: Muscle Energetics. Alan R. Liss, New York, 1989, pp 159–176Google Scholar
  27. 27.
    Pucar D, Dzeja PP, Bast P, Juranic N, Macura S, Terzic A: Cellular energetics in the preconditioned state: Protective role for phosphotransfer reactions captured by 18O-assisted 31P NMR. J Biol Chem 276: 44812–4489, 2001CrossRefPubMedGoogle Scholar
  28. 28.
    Steeghs K, Benders A, Oerlemans F, de Haan A, Heerschap A, Ruitenbeek W, Jost C, van Deursen J, Perryman B, Pette D, Bruckwilder M, Koudijs J, Jap P, Veerkamp J, Wieringa B: Altered Ca2+ responses in muscles with combined mitochondrial and cytosolic creatine kinase deficiencies. Cell 89: 93–103, 1997CrossRefPubMedGoogle Scholar
  29. 29.
    Janssen E, Dzeja PP, Oerlemans F, Simonetti AW, Heerschap A, de Haan A, Rush PS, Terjung RR, Wieringa B, Terzic A: Adenylate kinase 1 gene deletion disrupts muscle energetic economy despite metabolic rearrangement. EMBO J 19: 6371–6381, 2000CrossRefPubMedGoogle Scholar
  30. 30.
    Watchko JF, Daood MJ, Sieck GC, LaBella JJ, Ameredes BT, Koretsky AP, Wieringa B: Combined myofibrillar and mitochondrial creatine kinase deficiency impairs mouse diaphragm isotonic function. J Appl Physiol 82: 1416–1423, 1997CrossRefPubMedGoogle Scholar
  31. 31.
    Gorselink M, Drost MR, Coumans WA, van Kranenburg GP, Hesselink RP, van der Vusse GJ: Impaired muscular contractile performance and adenine nucleotide handling in creatine kinase-deficient mice. Am J Physiol 281: E619–E625, 2001PubMedGoogle Scholar
  32. 32.
    Crozatier B, Badoual T, Boehm E, Ennezat PV, Guenoun T, Su J, Veksler V, Hittinger L, Ventura-Clapier R: Role of creatine kinase in cardiac excitation-contraction coupling: Studies in creatine kinase-deficient mice. FASEB J 16: 653–660, 2002CrossRefPubMedGoogle Scholar
  33. 33.
    Saupe KW, Spindler M, Tian R, Ingwall JS: Impaired cardiac energetics in mice lacking muscle-specific isoenzymes of creatine kinase. Circ Res 82: 898–907, 1998PubMedGoogle Scholar
  34. 34.
    Jost CR, Van Der Zee CE, In 't Zandt HJ, Oerlemans F, Verheij M, Streijger F, Fransen J, Heerschap A, Cools AR, Wieringa B: Creatine kinase B-driven energy transfer in the brain is important for habituation and spatial learning behaviour, mossy fibre field size and determination of seizure susceptibility. Eur J Neurosci 15: 1692–1706, 2002CrossRefPubMedGoogle Scholar
  35. 35.
    Mahajan VB, Pai KS, Lau A, Cunningham DD: Creatine kinase, an ATP-generating enzyme, is required for thrombin receptor signaling to the cytoskeleton. Proc Natl Acad Sci USA 97: 12062–12067, 2000CrossRefPubMedGoogle Scholar
  36. 36.
    Pucar D, Janssen E, Dzeja PP, Juranic N, Macura S, Wieringa B, Terzic A: Compromised energetics in the adenylate kinase AK1 gene knockout heart under metabolic stress. J Biol Chem 275: 41424–41429, 2000CrossRefPubMedGoogle Scholar
  37. 37.
    Pucar D, Bast P, Gumina RJ, Lim L, Drahl C, Juranic N, Macura S, Janssen E, Wieringa B, Terzic A, Dzeja PP: Adenylate kinase AK1 knockout heart: Energetics and functional performance under ischemia-reperfusion. Am J Physiol 283: H776–H782, 2002Google Scholar
  38. 38.
    de Groof AJ, Oerlemans FT, Jost CR, Wieringa B: Changes in glycolytic network and mitochondrial design in creatine kinase-deficient muscles. Muscle Nerve 24: 1188–1196, 2001CrossRefPubMedGoogle Scholar
  39. 39.
    Ventura-Clapier R, Kuznetsov AV, d'Albis A, van Deursen J, Wieringa B, Veksler VI: Muscle creatine kinase-deficient mice. I. Alterations in myofibrillar function. J Biol Chem 270: 19914–19920, 1995CrossRefPubMedGoogle Scholar
  40. 40.
    Peusner L: In: Concepts in Bioenergetics. Englewood Cliffs, Prentice-Hall, 1974, pp 67–85Google Scholar
  41. 41.
    Zeleznikar RJ, Dzeja PP, Goldberg ND: Adenylate kinase-catalyzed phosphoryl transfer couples ATP utilization with its generation by glycolysis in intact muscle. J Biol Chem 270: 7311–7319, 1995CrossRefPubMedGoogle Scholar
  42. 42.
    Zeleznikar RJ, Goldberg ND: Kinetics and compartmentation of energy metabolism in intact skeletal muscle determined from 18O labeling of metabolite phosphoryls. J Biol Chem 266: 15110–15119, 1991PubMedGoogle Scholar
  43. 43.
    Seow CY, Stephens NL: Fatigue of mouse diaphragm muscle in isometric and isotonic contractions. J Appl Physiol 64: 2388–2393, 1988PubMedGoogle Scholar
  44. 44.
    Kekelidze T, Khait I, Togliatti A, Benzecry JM, Wieringa B, Holtzman D: Altered brain phosphocreatine and ATP regulation when mitochondrial creatine kinase is absent. J Neurosci Res 66: 866–872, 2001CrossRefPubMedGoogle Scholar
  45. 45.
    Spindler M, Niebler R, Remkes H, Horn M, Lanz T, Neubauer S: Mitochondrial creatine kinase is critically necessary for normal myocardial high-energy phosphate metabolism. Am J Physiol 283: H680–H687, 2002Google Scholar
  46. 46.
    Gerbitz KD, Gempel K, Brdiczka D: Mitochondria and diabetes. Genetic, biochemical, and clinical implications of the cellular energy circuit. Diabetes 45: 113–126, 1996PubMedGoogle Scholar
  47. 47.
    Dolder M, Wendt S, Wallimann T: Mitochondrial creatine kinase in contact sites: Interaction with porin and adenine nucleotide translocase, role in permeability transition and sensitivity to oxidative damage. Biol Signals Recep 10: 93–111, 2001CrossRefPubMedGoogle Scholar
  48. 48.
    Bandlow W, Strobel G, Zoglowek C, Oechsner U, Magdolen V: Yeast adenylate kinase is active simultaneously in mitochondria and cytoplasm and is required for non-fermentative growth. Eur J Biochem 178:451–457, 1988CrossRefPubMedGoogle Scholar
  49. 49.
    Roberts J, Aubert S, Gout E, Bligny R, Douce R: Cooperation and competition between adenylate kinase, nucleoside diphosphokinase, electron transport, and ATP synthase in plant mitochondria studied by 31P-nuclear magnetic resonance. Plant Physiol 113: 191–199, 1997PubMedGoogle Scholar
  50. 50.
    Ziegelhoffer-Mihalovicova B, Ziegelhoffer A, Ravingerova T, Kolar F, Jacob W, Tribulova N: Regulation of mitochondrial contact sites in neonatal, juvenile and diabetic hearts. Mol Cell Biochem 236: 37–44, 2002CrossRefPubMedGoogle Scholar
  51. 51.
    Kruiskamp MJ, van Vliet G, Nicolay K: 1H and 31P magnetization transfer studies of hindleg muscle in wild-type and creatine kinase-deficient mice. Magn Reson Med 43: 657–664, 2000CrossRefPubMedGoogle Scholar
  52. 52.
    Tian R, Christe ME, Spindler M, Hopkins JC, Halow J, Camacho SA, Ingwall JS: Role of MgADP in the development of diastolic dysfunction in the intact beating rat heart. J Clin Invest 99: 745–751, 1997PubMedGoogle Scholar
  53. 53.
    in 't Zandt HJ, Oerlemans F, Wieringa B, Heerschap A: Effects of ischemia on skeletal muscle energy metabolism in mice lacking creatine kinase monitored by in vivo 31P nuclear magnetic resonance spectroscopy. NMR Biomed 12: 327–334, 1999CrossRefPubMedGoogle Scholar
  54. 54.
    Saupe KW, Spindler M, Hopkins JC, Shen W, Ingwall JS: Kinetic, thermodynamic, and developmental consequences of deleting creatine kinase isoenzymes from the heart. Reaction kinetics of the creatine kinase isoenzymes in the intact heart. J Biol Chem 275: 19742–19746, 2000CrossRefPubMedGoogle Scholar
  55. 55.
    Goldbeter A: Patterns of spatiotemporal organization in an allosteric enzyme model. Proc Natl Acad Sci USA 70: 3255–3259, 1973PubMedGoogle Scholar
  56. 56.
    Mair T, Muller SC: Traveling NADH and proton waves during oscillatory glycolysis in vitro. J Biol Chem 271: 627–630, 1996CrossRefPubMedGoogle Scholar
  57. 57.
    Romashko DN, Marban E, O'Rourke B: Subcellular metabolic transients and mitochondrial redox waves in heart cells. Proc Natl Acad Sci USA 95: 1618–1623, 1998CrossRefPubMedGoogle Scholar
  58. 58.
    Stewart AK, Boyd CA, Vaughan-Jones RD: A novel role for carbonic anhydrase: Cytoplasmic pH gradient dissipation in mouse small intestinal enterocytes. J Physiol 516: 209–217, 1999CrossRefPubMedGoogle Scholar
  59. 59.
    Goldbeter A: Computational approaches to cellular rhythms. Nature 420: 238–245, 2002CrossRefPubMedGoogle Scholar
  60. 60.
    Kindzelskii AL, Petty HR: Apparent role of travelling metabolic waves in oxidant release by living neutrophils. Proc Natl Acad Sci USA 99: 9207–9212, 2002CrossRefPubMedGoogle Scholar
  61. 61.
    O'Rourke B, Ramza BM, Marban E: Oscillations of membrane current and excitability driven by metabolic oscillations in heart cells. Science 265: 962–966, 1994PubMedGoogle Scholar
  62. 62.
    LaBella JJ, Daood MJ, Koretsky AP, Roman BB, Sieck GC, Wieringa B, Watchko JF: Absence of myofibrillar creatine kinase and diaphragm isometric function during repetitive activation. J Appl Physiol 84: 1166–1173, 1998PubMedGoogle Scholar
  63. 63.
    Boehm E, Ventura-Clapier R, Mateo P, Lechene P, Veksler V: Glycolysis supports calcium uptake by the sarcoplasmic reticulum in skinned ventricular fibres of mice deficient in mitochondrial and cytosolic creatine kinase. J Mol Cell Cardiol 32: 891–902, 2000CrossRefPubMedGoogle Scholar
  64. 64.
    Kaasik A, Veksler V, Boehm E, Novotova M, Minajeva A, Ventura-Clapier R: Energetic crosstalk between organelles: architectural integration of energy production and utilization. Circ Res 89: 153–159, 2001PubMedGoogle Scholar
  65. 65.
    Sako EY, Kingsley-Hickman PB, From AH, Foker JE, Ugurbil K: ATP synthesis kinetics and mitochondrial function in the postischemic myocardium as studied by 31P NMR. J Biol Chem 263: 10600–10607, 1988PubMedGoogle Scholar
  66. 66.
    Pierce GN, Philipson KD: Binding of glycolytic enzymes to cardiac sarcolemmal and sarcoplasmic reticular membranes. J Biol Chem 260: 6862–6870, 1985PubMedGoogle Scholar
  67. 67.
    Ottaway JH, Mowbray J: The role of compartmentation in the control of glycolysis. Curr Top Cell Reg 12: 107–208, 1977Google Scholar
  68. 68.
    Parra J, Brdiczka D, Cusso R, Pette D: Enhanced catalytic activity of hexokinase by work-induced mitochondrial binding in fast-twitch muscle of rat. FEBS Lett 403: 279–282, 1997CrossRefPubMedGoogle Scholar
  69. 69.
    Ritov VB, Kelley DE: Hexokinase isozyme distribution in human skeletal muscle. Diabetes 50: 1253–1262, 2001PubMedGoogle Scholar
  70. 70.
    Wojtas K, Slepecky N, von Kalm L, Sullivan D: Flight muscle function in Drosophila requires colocalization of glycolytic enzymes. Mol Biol Cell 8: 1665–1675, 1997PubMedGoogle Scholar
  71. 71.
    Lange S, Auerbach D, McLoughlin P, Perriard E, Schafer BW, Perriard JC, Ehler E: Subcellular targeting of metabolic enzymes to titin in heart muscle may be mediated by DRAL/FHL-2. J Cell Sci 115: 4925–4936, 2002CrossRefPubMedGoogle Scholar
  72. 72.
    From AH, Zimmer SD, Michurski SP, Mohanakrishnan P, Ulstad VK, Thoma WJ, Ugurbil K: Regulation of the oxidative phosphorylation rate in the intact cell. Biochemistry 29: 3731–3743, 1990CrossRefPubMedGoogle Scholar
  73. 73.
    Wegmann G, Zanolla E, Eppenberger HM, Wallimann T: In situ compartmentation of creatine kinase in intact sarcomeric muscle: The actomyosin overlap zone as a molecular sieve. J Muscle Res Cell Motil 13: 420–435, 1992CrossRefPubMedGoogle Scholar
  74. 74.
    Busby SJ, Gadian DG, Radda GK, Richards RE, Seeley PJ: Phosphorus nuclear-magnetic-resonance studies of compartmentation in muscle. Biochem J 170: 103–114, 1978PubMedGoogle Scholar
  75. 75.
    Kemp GJ, Polgreen KE, Radda GK: Skeletal muscle Pi transport and cellular [Pi] studied in L6 myoblasts and rabbit muscle-membrane vesicles. Biochim Biophys Acta 1137: 10–18, 1992CrossRefPubMedGoogle Scholar
  76. 76.
    Bendahan D, Confort-Gouny S, Kozak-Reiss G, Cozzone PJ: Pi trapping in glycogenolytic pathway can explain transient Pi disappearance during recovery from muscular exercise. A 31P NMR study in the human. FEBS Lett 269: 402–405, 1990CrossRefPubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 2004

Authors and Affiliations

  • Petras P. Dzeja
    • 1
    • 2
  • Andre Terzic
    • 2
  • Be Wieringa
    • 3
  1. 1.Department of BiochemistryUniversity of MinnesotaMinneapolisUSA
  2. 2.Division of Cardiovascular Diseases, Departments of Medicine and Molecular Pharmacology and Experimental TherapeuticsMayo ClinicRochesterUSA
  3. 3.Department of Cell BiologyNCMLS University Medical Center, University of NijmegenThe Netherlands

Personalised recommendations