ADP Compartmentation Analysis Reveals Coupling between Pyruvate Kinase and ATPases in Heart Muscle

Cardiomyocytes have intracellular diffusion restrictions, which spatially compartmentalize ADP and ATP. However, the models that predict diffusion restrictions have used data sets generated in rat heart permeabilized fibers, where diffusion distances may be heterogeneous. This is avoided by using isolated, permeabilized cardiomyocytes. The aim of this work was to analyze the intracellular diffusion of ATP and ADP in rat permeabilized cardiomyocytes. To do this, we measured respiration rate, ATPase rate, and ADP concentration in the surrounding solution. The data were analyzed using mathematical models that reflect different levels of cell compartmentalization. In agreement with previous studies, we found significant diffusion restriction by the mitochondrial outer membrane and confirmed a functional coupling between mitochondria and a fraction of ATPases in the cell. In addition, our experimental data show that considerable activity of endogenous pyruvate kinase (PK) remains in the cardiomyocytes after permeabilization. A fraction of ATPases were inactive without ATP feedback by this endogenous PK. When analyzing the data, we were able to reproduce the measurements only with the mathematical models that include a tight coupling between the fraction of endogenous PK and ATPases. To our knowledge, this is the first time such a strong coupling of PK to ATPases has been demonstrated in permeabilized cardiomyocytes.

[1]  Marko Vendelin,et al.  Intracellular diffusion of adenosine phosphates is locally restricted in cardiac muscle , 2004, Molecular and Cellular Biochemistry.

[2]  M. L. Genova,et al.  Kinetics of integrated electron transfer in the mitochondrial respiratory chain: random collisions vs. solid state electron channeling. , 2007, American journal of physiology. Cell physiology.

[3]  K. Tepp,et al.  Ultra performance liquid chromatography analysis of adenine nucleotides and creatine derivatives for kinetic studies , 2009 .

[4]  A. Terzic,et al.  Cardiac system bioenergetics: metabolic basis of the Frank‐Starling law , 2006, The Journal of physiology.

[5]  M. Bilsen,et al.  Metabolic remodelling of the failing heart: beneficial or detrimental? , 2008 .

[6]  L. Kümmel,et al.  Ca, Mg-ATPase activity of permeabilised rat heart cells and its functional coupling to oxidative phosphorylation of the cells. , 1988, Cardiovascular research.

[7]  B. Brenner,et al.  Equilibration and exchange of fluorescently labeled molecules in skinned skeletal muscle fibers visualized by confocal microscopy. , 1995, Biophysical journal.

[8]  U. F. Rasmussen,et al.  Oxygen solubilities of media used in electrochemical respiration measurements. , 2003, Analytical biochemistry.

[9]  L. Becker,et al.  Ultrastructural Localization of Glycolytic Enzymes on Sarcoplasmic Reticulum Vesicles , 1998, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[10]  D. Segretain,et al.  Three dimensional arrangement of mitochondria and endoplasmic reticulum in the heart muscle fiber of the rat , 1981, The Anatomical record.

[11]  J. Hoerter,et al.  Functional development of the creatine kinase system in perinatal rabbit heart. , 1991, Circulation research.

[12]  Marko Vendelin,et al.  Three-dimensional mitochondrial arrangement in ventricular myocytes: from chaos to order. , 2006, American journal of physiology. Cell physiology.

[13]  D. Sackett,et al.  Regulation of respiration in brain mitochondria and synaptosomes: restrictions of ADP diffusion in situ, roles of tubulin, and mitochondrial creatine kinase , 2008, Molecular and Cellular Biochemistry.

[14]  J. Weiss,et al.  Cardiac ATP-sensitive K+ channels. Evidence for preferential regulation by glycolysis , 1989, The Journal of general physiology.

[15]  Marko Vendelin,et al.  Intracellular diffusion restrictions in isolated cardiomyocytes from rainbow trout , 2009, BMC Cell Biology.

[16]  J. Weiss,et al.  Thematic review series: Systems Biology Approaches to Metabolic and Cardiovascular Disorders. Network perspectives of cardiovascular metabolism Published, JLR Papers in Press, August 31, 2006. , 2006, Journal of Lipid Research.

[17]  A. Kuznetsov,et al.  Metabolic compartmentation and substrate channelling in muscle cells , 1994, Molecular and Cellular Biochemistry.

[18]  L. Kadaja,et al.  Comparative analysis of the bioenergetics of adult cardiomyocytes and nonbeating HL-1 cells: respiratory chain activities, glycolytic enzyme profiles, and metabolic fluxes. , 2009, Canadian journal of physiology and pharmacology.

[19]  A. Minajeva,et al.  Ca2+ uptake by cardiac sarcoplasmic reticulum ATPase in situ strongly depends on bound creatine kinase , 1996, Pflügers Archiv.

[20]  Johannes H G M van Beek,et al.  Glycolytic buffering affects cardiac bioenergetic signaling and contractile reserve similar to creatine kinase. , 2003, American journal of physiology. Heart and circulatory physiology.

[21]  D. Burkhoff,et al.  Metabolic inhibition in the perfused rat heart: evidence for glycolytic requirement for normal sodium homeostasis. , 1998, American journal of physiology. Heart and circulatory physiology.

[22]  Y. Usson,et al.  Heterogeneity of ADP diffusion and regulation of respiration in cardiac cells. , 2003, Biophysical journal.

[23]  Remo Guidieri.  Res , 1995, RES: Anthropology and Aesthetics.

[24]  Stefan Neubauer,et al.  The failing heart--an engine out of fuel. , 2007, The New England journal of medicine.

[25]  L. Becker,et al.  Functional coupling between glycolysis and sarcoplasmic reticulum Ca2+ transport. , 1995, Circulation research.

[26]  Marko Vendelin,et al.  Anisotropic diffusion of fluorescently labeled ATP in rat cardiomyocytes determined by raster image correlation spectroscopy , 2008, American journal of physiology. Cell physiology.

[27]  V. Saks,et al.  Early alteration of the control of mitochondrial function in myocardial ischemia. , 1997, Journal of molecular and cellular cardiology.

[28]  H. Gesser,et al.  Effects of hibernation on mitochondrial regulation and metabolic capacities in myocardium of painted turtle (Chrysemys picta). , 2004, Comparative biochemistry and physiology. Part A, Molecular & integrative physiology.

[29]  W. Coetzee,et al.  The Glycolytic Enzymes, Glyceraldehyde-3-phosphate Dehydrogenase, Triose-phosphate Isomerase, and Pyruvate Kinase Are Components of the KATP Channel Macromolecular Complex and Regulate Its Function* , 2005, Journal of Biological Chemistry.

[30]  H. Gesser,et al.  Intracellular compartmentation of cardiac fibres from rainbow trout and Atlantic cod--a general design of heart cells. , 2006, Biochimica et biophysica acta.

[31]  Marko Vendelin,et al.  Analysis of functional coupling: mitochondrial creatine kinase and adenine nucleotide translocase. , 2004, Biophysical journal.

[32]  S. Byrd,et al.  Functional coupling between sarcoplasmic-reticulum-bound creatine kinase and Ca(2+)-ATPase. , 1993, European journal of biochemistry.

[33]  B. Wieringa,et al.  Direct Evidence for the Control of Mitochondrial Respiration by Mitochondrial Creatine Kinase in Oxidative Muscle Cells in Situ * , 2000, The Journal of Biological Chemistry.

[34]  Ave Minajeva,et al.  Energetic Crosstalk Between Organelles: Architectural Integration of Energy Production and Utilization , 2001, Circulation research.

[35]  A. Kuznetsov,et al.  Different kinetics of the regulation of respiration in permeabilized cardiomyocytes and in HL-1 cardiac cells. Importance of cell structure/organization for respiration regulation. , 2006, Biochimica et biophysica acta.

[36]  D. Sackett,et al.  Tubulin binding blocks mitochondrial voltage-dependent anion channel and regulates respiration , 2008, Proceedings of the National Academy of Sciences.

[37]  C. Pison,et al.  Regulation of respiration controlled by mitochondrial creatine kinase in permeabilized cardiac cells in situ. Importance of system level properties. , 2009, Biochimica et biophysica acta.

[38]  Marko Vendelin,et al.  Diffusion Restrictions Surrounding Mitochondria: A Mathematical Model of Heart Muscle Fibers , 2009, Biophysical journal.

[39]  M. Vendelin,et al.  Regulation of mitochondrial respiration in heart cells analyzed by reaction-diffusion model of energy transfer. , 2000, American journal of physiology. Cell physiology.

[40]  K. Sahlin,et al.  Functional complexes of mitochondria with Ca,MgATPases of myofibrils and sarcoplasmic reticulum in muscle cells. , 2001, Biochimica et biophysica acta.