31P NMR Detection of Subcellular Creatine Kinase Fluxes in the Perfused Rat Heart

The subcellular fluxes of exchange of ATP and phosphocreatine (PCr) between mitochondria, cytosol, and ATPases were assessed by 31P NMR spectroscopy to investigate the pathways of energy transfer in a steady state beating heart. Using a combined analysis of four protocols of inversion magnetization transfer associated with biochemical data, three different creatine kinase (CK) activities were resolved in the rat heart perfused in isovolumic control conditions: (i) a cytosolic CK functioning at equilibrium (forward, Ff = PCr → ATP, and reverse flux,Fr = ATP → PCr = 3.3 mm·s−1), (ii) a CK localized in the vicinity of ATPases (MM-CK bound isoform) favoring ATP synthesis (Ff = 1.7 × Fr), and (iii) a mitochondrial CK displaced toward PCr synthesis (Ff = 0.3 and Fr = 2.6 mm·s−1). This study thus provides the first experimental evidence that the energy is carried from mitochondria to ATPases by PCr (i.e. CK shuttle) in the whole heart. In contrast, a single CK functioning at equilibrium was sufficient to describe the data when ATP synthesis was partly inhibited by cyanide (0.15 mm). In this case, ATP was directly transferred from mitochondria to cytosol suggesting that cardiac activity modified energy transfer pathways. Bioenergetic implications of the localization and activity of enzymes within myocardial cells are discussed. The subcellular fluxes of exchange of ATP and phosphocreatine (PCr) between mitochondria, cytosol, and ATPases were assessed by 31P NMR spectroscopy to investigate the pathways of energy transfer in a steady state beating heart. Using a combined analysis of four protocols of inversion magnetization transfer associated with biochemical data, three different creatine kinase (CK) activities were resolved in the rat heart perfused in isovolumic control conditions: (i) a cytosolic CK functioning at equilibrium (forward, Ff = PCr → ATP, and reverse flux,Fr = ATP → PCr = 3.3 mm·s−1), (ii) a CK localized in the vicinity of ATPases (MM-CK bound isoform) favoring ATP synthesis (Ff = 1.7 × Fr), and (iii) a mitochondrial CK displaced toward PCr synthesis (Ff = 0.3 and Fr = 2.6 mm·s−1). This study thus provides the first experimental evidence that the energy is carried from mitochondria to ATPases by PCr (i.e. CK shuttle) in the whole heart. In contrast, a single CK functioning at equilibrium was sufficient to describe the data when ATP synthesis was partly inhibited by cyanide (0.15 mm). In this case, ATP was directly transferred from mitochondria to cytosol suggesting that cardiac activity modified energy transfer pathways. Bioenergetic implications of the localization and activity of enzymes within myocardial cells are discussed. Two major roles have been attributed to the creatine kinase (CK, 1The abbreviations used are: CKcreatine kinaseANTadenine nucleotide translocatorATP1cytosolic ATPATP2mitochondrial ATPATP3ATP in the vicinity of the ATP-consuming sitesc.i.confidence intervalCrcreatinecyto-CKcytosolic CKEDPend diastolic pressureFfforward CK flux, unidirectional flux of ATP synthesis by CKFrreverse flux, unidirectional flux of PCr synthesis by CKi.m.s.intermembrane space between the outer and inner mitochondrial membraneinv- (inv-PCr or inv-ATP)inversion (of PCr or of γ-ATP), protocol of magnetization transfer experimentLVPleft ventricular systolic pressuremin-χ2minimal χ2, estimation of the quality of the adjustmentmito-CKmitochondrial CK isoformMM-bound CKMM creatine kinase isoform localized in the vicinity of ATP-consuming sitesPCrphosphocreatineQO2cardiac oxygen consumptionRPPrate pressure product (estimation of contractility)satPicontinuous saturation of inorganic phosphate resonanceSLsarcolemmaSRsarcoplasmic reticulumT1xspin lattice relaxation time of species x 1The abbreviations used are: CKcreatine kinaseANTadenine nucleotide translocatorATP1cytosolic ATPATP2mitochondrial ATPATP3ATP in the vicinity of the ATP-consuming sitesc.i.confidence intervalCrcreatinecyto-CKcytosolic CKEDPend diastolic pressureFfforward CK flux, unidirectional flux of ATP synthesis by CKFrreverse flux, unidirectional flux of PCr synthesis by CKi.m.s.intermembrane space between the outer and inner mitochondrial membraneinv- (inv-PCr or inv-ATP)inversion (of PCr or of γ-ATP), protocol of magnetization transfer experimentLVPleft ventricular systolic pressuremin-χ2minimal χ2, estimation of the quality of the adjustmentmito-CKmitochondrial CK isoformMM-bound CKMM creatine kinase isoform localized in the vicinity of ATP-consuming sitesPCrphosphocreatineQO2cardiac oxygen consumptionRPPrate pressure product (estimation of contractility)satPicontinuous saturation of inorganic phosphate resonanceSLsarcolemmaSRsarcoplasmic reticulumT1xspin lattice relaxation time of species x adenosine triphosphate creatine phosphotransferase, E.C. 2.7.3.2) reaction, which catalyzes the reversible exchange of high energy phosphate: PCr2−+MgADP−+H+ ⇄FrFf Cr+MgATP2−Equation 1 Due to its equilibrium constant favoring ATP synthesis, CK acts as a spatial and temporal buffer for ATP. In addition, due to specific intracellular localization, CK has been suggested to play a key role in energy transfer from sites of ATP production to ATPases by a phosphocreatine (PCr)-creatine (Cr)-CK shuttle. In the myocardial cell, half of the total CK activity is present in the cytosol (cyto-CK), and the other half is accounted for by MM and mitochondrial (mito-CK) isoforms located in the vicinity of other enzymes: a particulate MM-bound CK located in close vicinity to the ATPases of myofibrils, sarcoplasmic reticulum (SR), and sarcolemma (SL) and a mito-CK isoform that is close to the adenine nucleotide translocase (ANT) in the intermembrane space (i.m.s.) of mitochondria. The vicinity of CK with other enzymes was shown to influence enzyme kinetics both in vitro (1Sata M. Sugiura S. Yamashita H. Momomura S. Serizawa T. Circulation. 1996; 93: 310-317Crossref PubMed Scopus (32) Google Scholar, 2Arrio-Dupont M. Bechet J.J. D'Albis A. Eur. J. Biochem. 1992; 207: 951-955Crossref PubMed Scopus (14) Google Scholar) and in skinned fiber preparations (3Saks V.A. Kuznetsov A.V. Kupryianov V.V. Micelli M.V. Jacobus W.E. J. Biol. Chem. 1985; 260: 7757-7764Abstract Full Text PDF PubMed Google Scholar, 4Ventura-Clapier R. Mekhfi H. Vassort G. J. Gen. Physiol. 1987; 89: 815-837Crossref PubMed Scopus (61) Google Scholar). Mito-CK is the most obvious example of an exclusive localization in the restricted i.m.s. of the mitochondria where mito-CK octamers and their vicinity to ANT and porin have been proposed to efficiently channel energy from the mitochondria to cytosol (5Saks V.A. Khuchua Z.A. Vasilyeva E.V. Belikova O.Y. Kuznetsov A.V. Mol. Cell. Biochem. 1994; 133: 155-192Crossref PubMed Scopus (219) Google Scholar, 6Wyss M. Smeitink J. Wevers R.A. Wallimann T. Biophys. Biochim. Acta. 1992; 1102: 119-166Crossref PubMed Scopus (374) Google Scholar). The functional consequence of this localization was first evidenced in vitro where the activity of mito-CK changes the apparent affinity of respiration for externally added ADP in isolated mitochondria and saponin-skinned fibers (7Moreadith R.W. Jacobus W.E. J. Biol. Chem. 1982; 257: 899-905Abstract Full Text PDF PubMed Google Scholar, 8Jacobus W.E. Saks V.A. Arch. Biochem. Biophys. 1982; 219: 167-178Crossref PubMed Scopus (102) Google Scholar, 9Kay L. Nicolay K. Wieringa B. Saks V. Wallimann T. J. Biol. Chem. 2000; 275: 6937-6944Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). In other words there is a restriction of the outer mitochondrial membrane permeability to ADP. Although there is no physical membrane to restrict MM-CK isozyme in myofibrils, SR, or SL, the localization and activity of MM-CK modifies the kinetics of the myofibrillar ATPases (4Ventura-Clapier R. Mekhfi H. Vassort G. J. Gen. Physiol. 1987; 89: 815-837Crossref PubMed Scopus (61) Google Scholar), the SR Ca-ATPase (10Minajeva A. Ventura-Clapier R. Veksler V. Pflueg. Arch. 1996; 432: 904-912Crossref PubMed Scopus (64) Google Scholar), the Na,K-ATPase (11Saks V.A. Lipina N.V. Chernousova G.B. Sharov V.G. Smirnov V.N. Chazov E.I. Grosse R. Biochemistry (Mosc.). 1976; 41: 1701-1709Google Scholar), and SL ionic channels (12Crawford R.M. Ranki H.J. Botting C.H. Budas G.R. Jovanovic A. FASEB J. 2002; 16: 102-104Crossref PubMed Scopus (122) Google Scholar). Thus, in the highly organized structure of an adult myocardial cell, the localization of enzymes at distinct subcellular sites may be regulated in different ways, play distinct roles, and fulfill specialized functions. This localization may contribute to cardiac efficiency by playing a crucial role in energy transfer (for review, see Refs. 13Wallimann T. Wyss M. Brdiczka D. Nicolay K. Eppenberger H.M. Biochem. J. 1992; 281: 21-40Crossref PubMed Scopus (1573) Google Scholar and 14Saks V.A. Ventura-Clapier R. Aliev M.K. Biophys. Biochim. Acta. 1996; 1274: 81-88Crossref PubMed Scopus (109) Google Scholar). The complex interplay of subcellular energy exchanges emphasizes the need for specific information on the subcellular energetic exchanges in the whole heart. Several mathematical models have been proposed to evaluate the subcellular CK fluxes in the whole organ (15Aliev M.K. Saks V.A. Biophys. J. Full Text PDF PubMed Scopus Google Scholar, Mol. Cell. Biochem. PubMed Google Scholar, M. Saks V. J. Physiol. 2000; PubMed Google Scholar, J. Physiol. 2000; PubMed Google Scholar). on of the structure of the exchange the of the CK kinetics from in vitro data, a restriction in ADP at the outer mitochondrial and was to an experimental that on creatine kinase adenine nucleotide cytosolic ATP mitochondrial ATP ATP in the vicinity of the ATP-consuming sites creatine cytosolic CK diastolic pressure CK flux, unidirectional flux of ATP synthesis by CK reverse flux, unidirectional flux of PCr synthesis by CK intermembrane space between the outer and inner mitochondrial membrane inversion (of PCr or of γ-ATP), protocol of magnetization transfer ventricular systolic pressure χ2, estimation of the quality of the mitochondrial CK isoform MM creatine kinase isoform localized in the vicinity of ATP-consuming sites phosphocreatine cardiac oxygen pressure product (estimation of saturation of inorganic phosphate sarcolemma sarcoplasmic reticulum lattice relaxation time of species x creatine kinase adenine nucleotide cytosolic ATP mitochondrial ATP ATP in the vicinity of the ATP-consuming sites creatine cytosolic CK diastolic pressure CK flux, unidirectional flux of ATP synthesis by CK reverse flux, unidirectional flux of PCr synthesis by CK intermembrane space between the outer and inner mitochondrial membrane inversion (of PCr or of γ-ATP), protocol of magnetization transfer ventricular systolic pressure χ2, estimation of the quality of the mitochondrial CK isoform MM creatine kinase isoform localized in the vicinity of ATP-consuming sites phosphocreatine cardiac oxygen pressure product (estimation of saturation of inorganic phosphate sarcolemma sarcoplasmic reticulum lattice relaxation time of species x to intracellular has been in NMR analysis of CK kinetics in the whole by Wallimann T. 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Full Text Full Text PDF PubMed Scopus Google Scholar). 1 the of analysis used to the of exchange the NMR the time of and PCr magnetization in of the four inversion protocols were combined in a single of Several of exchange of the species the pathways of energy synthesis and were was no a of exchange an of with the were the of magnetization by the four inversion protocols were from the specific exchange and data were The of relaxation ATP and PCr and of MM-bound when were to the of and PCr of the four inversion The quality of the was by the where and are the experimental data for the from 1 to with their and the and are the of the as when its and the were The of exchange was by of the as to the the of ATP, and as assessed by NMR and the of the and reverse fluxes state of as in a steady state organ as Biophys. J. Full Text Full Text PDF PubMed Scopus Google Scholar). of in both and reverse fluxes of the the were (i) the of mitochondrial ATP synthesis flux as from the oxygen in the V. B. 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PubMed Scopus Google and of ATP, and in of pressure and in in the of the = are is in the pressure product = × heart is in and is in of ATP synthesis from was for the control and a of and an intracellular of of in a ATP, and in of pressure and in in the of the = are is in the pressure product = × heart is in and is in of ATP synthesis from was for the control and a of and an intracellular of of of a protocol of and the time of PCr and magnetization are shown for a control heart a and The time of PCr and magnetization of four to perfused for inversion was in a single of data for analysis for the control and for the the of PCr and ATP magnetization was for the of exchange in from three to subcellular The was first to as a of 1 the energetic exchange as a CK flux and an exchange of ATP with a mitochondrial ATP was directly with cytosolic ATP, or mitochondrial CK three specific CK fluxes and with three ATP the ATP at the of the myofibrils, SR, and The of the of exchange shown in to a structure of exchange the NMR In the a between ATP, and the was = The of a mitochondrial ATP directly with cytosolic ATP the of = In the = = when with PCr the mito-CK which the of three ATP cytosolic mitochondrial and ATP bound close to myofibrils, SR, and and their exchange three CK the data = = Thus, in control the of three distinct CK the the NMR experimental the for the control in the exchange The CK flux was in the by NMR protocols at V. B. J. Physiol. Scholar, K. M. R. S. PubMed Scopus Google Scholar). a to to when CK subcellular localization was in the of ATP and PCr were by the which from to for PCr and to for ATP, were in with The most was the in subcellular CK kinetics on their mito-CK was and no mitochondrial CK flux be In from the analysis as exchange the flux Ff of ATP synthesis was (Ff = 0.3 = with the flux of PCr synthesis = 2.6 which accounted for of the total reverse CK the at the of MM-bound both and reverse fluxes were The ATP synthesis (Ff was The ATP MM-bound CK to = of total ATP. CK was at equilibrium and accounted for of the total CK in for the control to the flux PCr → and Fr to → PCr of = total or reverse CK of exchange the of the are shown as the and in to The exchange was in The ATP, and steady state for of Ff and Fr CK flux = from oxygen and of mitochondrial = and = of total in In which the data the to of total ATP = cytosolic PCr mitochondrial PCr in a Ff to the flux PCr → and Fr to → PCr of = total or reverse CK of exchange the of the are shown as the and in to The exchange was in The ATP, and steady state for of Ff and Fr CK flux = from oxygen and of mitochondrial = and = of total in In which the data the to of total ATP = cytosolic PCr mitochondrial PCr the of the for control which the to a was the of analysis = = The quality of was for a exchange of mitochondrial to cytosolic ATP = = In with the control the of a mito-CK flux that of an MM-bound CK the the analysis the of the of MM-bound CK and mito-CK to the CK the was their flux the of energetic exchanges in the cyanide the of different the that a to the of ATP synthesis, of respiration ATP this ATP synthesis flux in an of the flux of ATP evaluate the influence of this an cytosolic ATP synthesis → to the exchange the ATP synthesis from to by the of → flux in a of the from a of in the of this to This that in of mitochondrial ATP synthesis by to ATP synthesis was In the exchange the experimental of of the relaxation = 1.7 and = for and and = for were to in the control for an in = and reverse CK fluxes = Although between CK flux and the CK flux, in a to be in the control when CK was in the The CK flux to the CK flux in the by saturation transfer V. 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The of creatine in control and the of mito-CK activity in as a that the of energy transfer between mitochondria and sites of ATP with cardiac a of energy transfer in both experimental In control the of mito-CK from equilibrium in a flux of PCr synthesis, and PCr was the species from mitochondria to cytosol and ATP-consuming sites an exclusive flux of ATP between mitochondria and cytosol in the of mito-CK activity was as the of exchange shown in this the that both PCr and ATP from mitochondria in myocardial energy was to both pathways due to the in to evaluate this by In as a a of ATP from mitochondria to cytosol at the of PCr and the influence of this on the quality of the transfer of → from to of mitochondrial ATP synthesis, exchanges of ATP between were was by a exchange of → to or by a activity of → and → to cytosol and cytosol to an exclusive → the quality of the Although this the of a ATP from mitochondria to cytosol, its influence on the of the data was in with a major role of mito-CK in the transfer of energy in the control heart. cardiac was by of ATP synthesis, a CK functioning at equilibrium was sufficient to describe the NMR an mitochondrial ATP by the adenine nucleotide translocase in i.m.s. was directly to cytosol by a flux, to the ANT This from a transfer of energy by phosphocreatine in control isovolumic of to the of ATP in that the pathways of energy transfer on the cardiac high cardiac activity the shuttle. that NMR spectroscopy has the to and subcellular exchanges in the whole organ the of analysis is In control that subcellular have different on their localization Biophys. 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