Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2001 Jun 1;356(Pt 2):643–657. doi: 10.1042/0264-6021:3560643

Intracellular energetic units in red muscle cells.

V A Saks 1, T Kaambre 1, P Sikk 1, M Eimre 1, E Orlova 1, K Paju 1, A Piirsoo 1, F Appaix 1, L Kay 1, V Regitz-Zagrosek 1, E Fleck 1, E Seppet 1
PMCID: PMC1221880  PMID: 11368796

Abstract

The kinetics of regulation of mitochondrial respiration by endogenous and exogenous ADP in muscle cells in situ was studied in skinned cardiac and skeletal muscle fibres. Endogenous ADP production was initiated by addition of MgATP; under these conditions the respiration rate and ADP concentration in the medium were dependent on the calcium concentration, and 70-80% of maximal rate of respiration was achieved at ADP concentration below 20 microM in the medium. In contrast, when exogenous ADP was added, maximal respiration rate was observed only at millimolar concentrations. An exogenous ADP-consuming system consisting of pyruvate kinase (PK; 20-40 units/ml) and phosphoenolpyruvate (PEP; 5 mM), totally suppressed respiration activated by exogenous ADP, but the respiration maintained by endogenous ADP was not suppressed by more than 20-40%. Creatine (20 mM) further activated respiration in the presence of ATP and PK+PEP. Short treatment with trypsin (50-500 nM for 5 min) decreased the apparent K(m) for exogenous ADP from 300-350 microM to 50-60 microM, increased inhibition of respiration by PK+PEP system up to 70-80%, with no changes in MgATPase activity and maximal respiration rates. Electron-microscopic observations showed detachment of mitochondria and disordering of the regular structure of the sarcomere after trypsin treatment. Two-dimensional electrophoresis revealed a group of at least seven low-molecular-mass proteins in cardiac skinned fibres which were very sensitive to trypsin and not present in glycolytic fibres, which have low apparent K(m) for exogenous ADP. It is concluded that, in oxidative muscle cells, mitochondria are incorporated into functional complexes ('intracellular energetic units') with adjacent ADP-producing systems in myofibrils and in sarcoplasmic reticulum, probably due to specific interaction with cytoskeletal elements responsible for mitochondrial distribution in the cell. It is suggested that these complexes represent the basic pattern of organization of muscle-cell energy metabolism.

Full Text

The Full Text of this article is available as a PDF (633.2 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Altschuld R., Hohl C., Ansel A., Brierley G. P. Compartmentation of K+ in isolated adult rat heart cells. Arch Biochem Biophys. 1981 Jun;209(1):175–184. doi: 10.1016/0003-9861(81)90270-8. [DOI] [PubMed] [Google Scholar]
  2. Balaban R. S., Kantor H. L., Katz L. A., Briggs R. W. Relation between work and phosphate metabolite in the in vivo paced mammalian heart. Science. 1986 May 30;232(4754):1121–1123. doi: 10.1126/science.3704638. [DOI] [PubMed] [Google Scholar]
  3. Bernardi P., Scorrano L., Colonna R., Petronilli V., Di Lisa F. Mitochondria and cell death. Mechanistic aspects and methodological issues. Eur J Biochem. 1999 Sep;264(3):687–701. doi: 10.1046/j.1432-1327.1999.00725.x. [DOI] [PubMed] [Google Scholar]
  4. Bessman S. P., Geiger P. J. Transport of energy in muscle: the phosphorylcreatine shuttle. Science. 1981 Jan 30;211(4481):448–452. doi: 10.1126/science.6450446. [DOI] [PubMed] [Google Scholar]
  5. Boyer P. D. The ATP synthase--a splendid molecular machine. Annu Rev Biochem. 1997;66:717–749. doi: 10.1146/annurev.biochem.66.1.717. [DOI] [PubMed] [Google Scholar]
  6. Brandes R., Bers D. M. Analysis of the mechanisms of mitochondrial NADH regulation in cardiac trabeculae. Biophys J. 1999 Sep;77(3):1666–1682. doi: 10.1016/S0006-3495(99)77014-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Capaldi R. A. Arrangement of proteins in the mitochondrial inner membrane. Biochim Biophys Acta. 1982 Nov 30;694(3):291–306. doi: 10.1016/0304-4157(82)90009-0. [DOI] [PubMed] [Google Scholar]
  8. Chase T., Jr, Shaw E. Comparison of the esterase activities of trypsin, plasmin, and thrombin on guanidinobenzoate esters. Titration of the enzymes. Biochemistry. 1969 May;8(5):2212–2224. doi: 10.1021/bi00833a063. [DOI] [PubMed] [Google Scholar]
  9. Cheung C. W., Cohen N. S., Raijman L. Channeling of urea cycle intermediates in situ in permeabilized hepatocytes. J Biol Chem. 1989 Mar 5;264(7):4038–4044. [PubMed] [Google Scholar]
  10. Duchen M. R. Contributions of mitochondria to animal physiology: from homeostatic sensor to calcium signalling and cell death. J Physiol. 1999 Apr 1;516(Pt 1):1–17. doi: 10.1111/j.1469-7793.1999.001aa.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Duchen M. R., Leyssens A., Crompton M. Transient mitochondrial depolarizations reflect focal sarcoplasmic reticular calcium release in single rat cardiomyocytes. J Cell Biol. 1998 Aug 24;142(4):975–988. doi: 10.1083/jcb.142.4.975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Eggleton P., Elsden S. R., Gough N. The estimation of creatine and of diacetyl. Biochem J. 1943;37(5):526–529. doi: 10.1042/bj0370526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fiolet J. W., Baartscheer A., Schumacher C. A. Intracellular [Ca2+] and Vo2 after manipulation of the free-energy of the Na+/Ca(2+)-exchanger in isolated rat ventricular myocytes. J Mol Cell Cardiol. 1995 Aug;27(8):1513–1525. doi: 10.1016/s0022-2828(95)90260-0. [DOI] [PubMed] [Google Scholar]
  14. Gellerich F., Saks V. A. Control of heart mitochondrial oxygen consumption by creatine kinase: the importance of enzyme localization. Biochem Biophys Res Commun. 1982 Apr 29;105(4):1473–1481. doi: 10.1016/0006-291x(82)90954-8. [DOI] [PubMed] [Google Scholar]
  15. Gudbjarnason S., Mathes P., Ravens K. G. Functional compartmentation of ATP and creatine phosphate in heart muscle. J Mol Cell Cardiol. 1970 Sep;1(3):325–339. doi: 10.1016/0022-2828(70)90009-x. [DOI] [PubMed] [Google Scholar]
  16. Higuchi H. Changes in contractile properties with selective digestion of connectin (titin) in skinned fibers of frog skeletal muscle. J Biochem. 1992 Mar;111(3):291–295. doi: 10.1093/oxfordjournals.jbchem.a123752. [DOI] [PubMed] [Google Scholar]
  17. Jungblut P., Otto A., Zeindl-Eberhart E., Plessner K. P., Knecht M., Regitz-Zagrosek V., Fleck E., Wittmann-Liebold B. Protein composition of the human heart: the construction of a myocardial two-dimensional electrophoresis database. Electrophoresis. 1994 May;15(5):685–707. doi: 10.1002/elps.1150150197. [DOI] [PubMed] [Google Scholar]
  18. Katz L. A., Koretsky A. P., Balaban R. S. Activation of dehydrogenase activity and cardiac respiration: a 31P-NMR study. Am J Physiol. 1988 Jul;255(1 Pt 2):H185–H188. doi: 10.1152/ajpheart.1988.255.1.H185. [DOI] [PubMed] [Google Scholar]
  19. Kay L., Li Z., Mericskay M., Olivares J., Tranqui L., Fontaine E., Tiivel T., Sikk P., Kaambre T., Samuel J. L. Study of regulation of mitochondrial respiration in vivo. An analysis of influence of ADP diffusion and possible role of cytoskeleton. Biochim Biophys Acta. 1997 Nov 10;1322(1):41–59. doi: 10.1016/s0005-2728(97)00071-6. [DOI] [PubMed] [Google Scholar]
  20. Kay L., Nicolay K., Wieringa B., Saks V., Wallimann T. Direct evidence for the control of mitochondrial respiration by mitochondrial creatine kinase in oxidative muscle cells in situ. J Biol Chem. 2000 Mar 10;275(10):6937–6944. doi: 10.1074/jbc.275.10.6937. [DOI] [PubMed] [Google Scholar]
  21. Khuchua Z., Belikova Y., Kuznetsov A. V., Gellerich F. N., Schild L., Neumann H. W., Kunz W. S. Caffeine and Ca2+ stimulate mitochondrial oxidative phosphorylation in saponin-skinned human skeletal muscle fibers due to activation of actomyosin ATPase. Biochim Biophys Acta. 1994 Dec 30;1188(3):373–379. doi: 10.1016/0005-2728(94)90058-2. [DOI] [PubMed] [Google Scholar]
  22. Knecht M., Regitz-Zagrosek V., Pleissner K. P., Emig S., Jungblut P., Hildebrandt A., Fleck E. Dilated cardiomyopathy: computer-assisted analysis of endomyocardial biopsy protein patterns by two-dimensional gel electrophoresis. Eur J Clin Chem Clin Biochem. 1994 Aug;32(8):615–624. doi: 10.1515/cclm.1994.32.8.615. [DOI] [PubMed] [Google Scholar]
  23. Knecht M., Regitz-Zagrosek V., Pleissner K. P., Jungblut P., Steffen C., Hildebrandt A., Fleck E. Characterization of myocardial protein composition in dilated cardiomyopathy by two-dimensional gel electrophoresis. Eur Heart J. 1994 Dec;15 (Suppl 500):37–44. doi: 10.1093/eurheartj/15.suppl_d.37. [DOI] [PubMed] [Google Scholar]
  24. Korzeniewski B. Regulation of ATP supply during muscle contraction: theoretical studies. Biochem J. 1998 Mar 15;330(Pt 3):1189–1195. doi: 10.1042/bj3301189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kuznetsov A. V., Tiivel T., Sikk P., Kaambre T., Kay L., Daneshrad Z., Rossi A., Kadaja L., Peet N., Seppet E. Striking differences between the kinetics of regulation of respiration by ADP in slow-twitch and fast-twitch muscles in vivo. Eur J Biochem. 1996 Nov 1;241(3):909–915. doi: 10.1111/j.1432-1033.1996.00909.x. [DOI] [PubMed] [Google Scholar]
  26. Kümmel L. Ca, Mg-ATPase activity of permeabilised rat heart cells and its functional coupling to oxidative phosphorylation of the cells. Cardiovasc Res. 1988 May;22(5):359–367. doi: 10.1093/cvr/22.5.359. [DOI] [PubMed] [Google Scholar]
  27. Lea P. J., Temkin R. J., Freeman K. B., Mitchell G. A., Robinson B. H. Variations in mitochondrial ultrastructure and dynamics observed by high resolution scanning electron microscopy (HRSEM). Microsc Res Tech. 1994 Mar 1;27(4):269–277. doi: 10.1002/jemt.1070270402. [DOI] [PubMed] [Google Scholar]
  28. Mannella C. A., Buttle K., Rath B. K., Marko M. Electron microscopic tomography of rat-liver mitochondria and their interaction with the endoplasmic reticulum. Biofactors. 1998;8(3-4):225–228. doi: 10.1002/biof.5520080309. [DOI] [PubMed] [Google Scholar]
  29. McCormack J. G., Halestrap A. P., Denton R. M. Role of calcium ions in regulation of mammalian intramitochondrial metabolism. Physiol Rev. 1990 Apr;70(2):391–425. doi: 10.1152/physrev.1990.70.2.391. [DOI] [PubMed] [Google Scholar]
  30. Milner D. J., Mavroidis M., Weisleder N., Capetanaki Y. Desmin cytoskeleton linked to muscle mitochondrial distribution and respiratory function. J Cell Biol. 2000 Sep 18;150(6):1283–1298. doi: 10.1083/jcb.150.6.1283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Müller E. C., Thiede B., Zimny-Arndt U., Scheler C., Prehm J., Müller-Werdan U., Wittmann-Liebold B., Otto A., Jungblut P. High-performance human myocardial two-dimensional electrophoresis database: edition 1996. Electrophoresis. 1996 Nov;17(11):1700–1712. doi: 10.1002/elps.1150171107. [DOI] [PubMed] [Google Scholar]
  32. Neely J. R., Grotyohann L. W. Role of glycolytic products in damage to ischemic myocardium. Dissociation of adenosine triphosphate levels and recovery of function of reperfused ischemic hearts. Circ Res. 1984 Dec;55(6):816–824. doi: 10.1161/01.res.55.6.816. [DOI] [PubMed] [Google Scholar]
  33. Ogata T., Yamasaki Y. Ultra-high-resolution scanning electron microscopy of mitochondria and sarcoplasmic reticulum arrangement in human red, white, and intermediate muscle fibers. Anat Rec. 1997 Jun;248(2):214–223. doi: 10.1002/(SICI)1097-0185(199706)248:2<214::AID-AR8>3.0.CO;2-S. [DOI] [PubMed] [Google Scholar]
  34. Post J. A., Langer G. A., Op den Kamp J. A., Verkleij A. J. Phospholipid asymmetry in cardiac sarcolemma. Analysis of intact cells and 'gas-dissected' membranes. Biochim Biophys Acta. 1988 Aug 18;943(2):256–266. doi: 10.1016/0005-2736(88)90557-3. [DOI] [PubMed] [Google Scholar]
  35. Rappaport L., Oliviero P., Samuel J. L. Cytoskeleton and mitochondrial morphology and function. Mol Cell Biochem. 1998 Jul;184(1-2):101–105. [PubMed] [Google Scholar]
  36. Rizzuto R., Pinton P., Carrington W., Fay F. S., Fogarty K. E., Lifshitz L. M., Tuft R. A., Pozzan T. Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses. Science. 1998 Jun 12;280(5370):1763–1766. doi: 10.1126/science.280.5370.1763. [DOI] [PubMed] [Google Scholar]
  37. Romashko D. N., Marban E., O'Rourke B. Subcellular metabolic transients and mitochondrial redox waves in heart cells. Proc Natl Acad Sci U S A. 1998 Feb 17;95(4):1618–1623. doi: 10.1073/pnas.95.4.1618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. SCHOLLMEYER P., KLINGENBERG M. [On the cytochrome content of animal tissue]. Biochem Z. 1962;335:426–439. [PubMed] [Google Scholar]
  39. Saks V. A., Belikova Y. O., Kuznetsov A. V. In vivo regulation of mitochondrial respiration in cardiomyocytes: specific restrictions for intracellular diffusion of ADP. Biochim Biophys Acta. 1991 Jul 8;1074(2):302–311. doi: 10.1016/0304-4165(91)90168-g. [DOI] [PubMed] [Google Scholar]
  40. Saks V. A., Khuchua Z. A., Vasilyeva E. V., Belikova OYu, Kuznetsov A. V. Metabolic compartmentation and substrate channelling in muscle cells. Role of coupled creatine kinases in in vivo regulation of cellular respiration--a synthesis. Mol Cell Biochem. 1994 Apr-May;133-134:155–192. doi: 10.1007/BF01267954. [DOI] [PubMed] [Google Scholar]
  41. Saks V. A., Kuznetsov A. V., Khuchua Z. A., Vasilyeva E. V., Belikova J. O., Kesvatera T., Tiivel T. Control of cellular respiration in vivo by mitochondrial outer membrane and by creatine kinase. A new speculative hypothesis: possible involvement of mitochondrial-cytoskeleton interactions. J Mol Cell Cardiol. 1995 Jan;27(1):625–645. doi: 10.1016/s0022-2828(08)80056-9. [DOI] [PubMed] [Google Scholar]
  42. Saks V. A., Tiivel T., Kay L., Novel-Chaté V., Daneshrad Z., Rossi A., Fontaine E., Keriel C., Leverve X., Ventura-Clapier R. On the regulation of cellular energetics in health and disease. Mol Cell Biochem. 1996 Jul-Aug;160-161:195–208. doi: 10.1007/BF00240050. [DOI] [PubMed] [Google Scholar]
  43. Saks V. A., Vasil'eva E., Belikova YuO, Kuznetsov A. V., Lyapina S., Petrova L., Perov N. A. Retarded diffusion of ADP in cardiomyocytes: possible role of mitochondrial outer membrane and creatine kinase in cellular regulation of oxidative phosphorylation. Biochim Biophys Acta. 1993 Sep 13;1144(2):134–148. doi: 10.1016/0005-2728(93)90166-d. [DOI] [PubMed] [Google Scholar]
  44. Saks V. A., Veksler V. I., Kuznetsov A. V., Kay L., Sikk P., Tiivel T., Tranqui L., Olivares J., Winkler K., Wiedemann F. Permeabilized cell and skinned fiber techniques in studies of mitochondrial function in vivo. Mol Cell Biochem. 1998 Jul;184(1-2):81–100. [PubMed] [Google Scholar]
  45. Smirnova E., Shurland D. L., Ryazantsev S. N., van der Bliek A. M. A human dynamin-related protein controls the distribution of mitochondria. J Cell Biol. 1998 Oct 19;143(2):351–358. doi: 10.1083/jcb.143.2.351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Srere P. A. Macromolecular interactions: tracing the roots. Trends Biochem Sci. 2000 Mar;25(3):150–153. doi: 10.1016/s0968-0004(00)01550-4. [DOI] [PubMed] [Google Scholar]
  47. Stapulionis R., Deutscher M. P. A channeled tRNA cycle during mammalian protein synthesis. Proc Natl Acad Sci U S A. 1995 Aug 1;92(16):7158–7161. doi: 10.1073/pnas.92.16.7158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Vendelin M., Kongas O., Saks V. Regulation of mitochondrial respiration in heart cells analyzed by reaction-diffusion model of energy transfer. Am J Physiol Cell Physiol. 2000 Apr;278(4):C747–C764. doi: 10.1152/ajpcell.2000.278.4.C747. [DOI] [PubMed] [Google Scholar]
  49. Wallimann T., Wyss M., Brdiczka D., Nicolay K., Eppenberger H. M. 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. 1992 Jan 1;281(Pt 1):21–40. doi: 10.1042/bj2810021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Williamson J. R., Ford C., Illingworth J., Safer B. Coordination of citric acid cycle activity with electron transport flux. Circ Res. 1976 May;38(5 Suppl 1):I39–I51. [PubMed] [Google Scholar]
  51. Yaffe M. P. The machinery of mitochondrial inheritance and behavior. Science. 1999 Mar 5;283(5407):1493–1497. doi: 10.1126/science.283.5407.1493. [DOI] [PubMed] [Google Scholar]
  52. de Graaf R. A., van Kranenburg A., Nicolay K. In vivo (31)P-NMR diffusion spectroscopy of ATP and phosphocreatine in rat skeletal muscle. Biophys J. 2000 Apr;78(4):1657–1664. doi: 10.1016/S0006-3495(00)76717-8. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES