Skip to main content
SpringerLink
Log in

The contractile behaviour of EGTA- and detergent-treated heart muscle

  • Papers
  • Published:
Journal of Muscle Research & Cell Motility Aims and scope Submit manuscript

Summary

Tension responses of rat ventricular trabeculae subjected to successive ‘treatment’ with EGTA and Triton X-100 are described in order to investigate the effects of chemical ‘skinning’ techniques. In some preparations the alkaloid saponin was also used before Triton. Ultrastructural evidence is cited that the ‘EGTA-treatment’ fails to render cells ‘hyperpermeable’, i.e. freely permeable to small ions, whereas both saponin and Triton do so. In this paper we show that contractile responses like those described previously for the ‘EGTA-treated’ tissue can be obtained. However, more detailed examination shows that such behaviour is quantitatively distinct from that of conventionally skinned fibres in a way that is incompatible with the notion of ‘hyperpermeability’. The Ca-sensitivity after treatment with either EGTA, saponin or Triton is identical in our hands. However, this is not explained by free access of Ca (and EGTA) to the intracellular space in the EGTA-treated preparation: contractures develop with very different time courses, being fastest after Triton and only marginally slower when first exposed to saponin but a factor of five times slower after ‘EGTA-treatment’ alone. This applies to contractures evoked direct from Ca2+ concentration ⋍ 10−9 m to the test Ca2+ concentration at constant total buffer concentration.

‘EGTA-treated’ fibres develop tension when ATP or creatine phosphate (CrP) are removed from the bath. However, responses to ADP and to CrP changes persist with millimolar levels of ATP present, quite unlike the Triton-skinned muscle. Exposure to each of a variety of solutions for 24h produce preparations showing similar behaviour: whatever the explanation for the EGTA-‘skinning’ phenomenon it is not dependent upon low bathing Ca2+ concentration. On the basis of the functional characteristics described here, and the structural results cited, we conclude that the cell membrane continues to function as a selective permeability barrier after ‘EGTA-treatment’: this treatment does not produce a model of a selectively ‘skinned’ cardiac cell.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • ABBOTT, R. H. & LEECH, A. R. (1973) Persistence of adenylate kinase and other enzymes in glycerol extracted muscle.Pflügers Arch. 344, 233–43.

    Google Scholar 

  • ABOOD, L. G., KOKETSU, K. & MIYAMOTO, S. (1962) Outflux of various phosphates during membrane depolarisation of excitable tissues.Am. J. Physiol. 202, 469–74.

    Google Scholar 

  • ALMERS, W., FINK, R. & PALADE, P. T. (1981) Calcium deprivation in frog muscle tubules: the decline of calcium current under maintained depolarization.J. Physiol. 312, 177–208.

    Google Scholar 

  • ARMSTRONG, C. M., BEZANILLA, F. M. & HOROWICZ, P. (1972) Twitches in the presence of ethylene glycol bis (beta-amino-ethyl ether)-N,N′-tetraacetic acid.Biochim. biophys. Acta. 267, 605–8.

    Google Scholar 

  • ASHLEY, C. C. & MOISESCU, D. G. (1977) Effect of changing the composition of the bathing solution upon the isometric tension-pCa relationship in bundles of crustacean myofibrils.J. Physiol. 207, 627–52.

    Google Scholar 

  • BARRETT, J. N. & BARRETT, E. F. (1978) Excitation-contraction coupling in skeletal muscle: blockade by high extracellular concentrations of calcium buffers.Science 200, 1270–2.

    Google Scholar 

  • BEELER, G. W. & REUTER, H. (1977) Reconstruction of the action potential of ventricular myocardial fibres.J. Physiol. 268, 177–210.

    Google Scholar 

  • BENNETT, J. P., COCKCROFT, S. & GOMPERTS, B. D. (1982) Rat mast cells permeabilized with ATP secrete histamine in response to calcium ions buffered in the micromolar range.J. Physiol. 317, 335–46.

    Google Scholar 

  • BERS, D. (1982) A simple method for the accurate determination of free Ca in Ca-EGTA solutions.Am. J. Physiol. 242, C404–8.

    Google Scholar 

  • BLINKS, J. R., PRENDERGAST, F. G. & ALLEN, D. G. (1976) Photoproteins as biological indicators.Pharmac. Rev. 28, 1–93.

    Google Scholar 

  • BLINKS, J. R., WIER, W. G., HESS, P. & PRENDERGAST, F. G. (1982) Measurement of Ca2+ concentration in living cells.Prog. Biophys. molec. Biol. 40, 1–114.

    Google Scholar 

  • BOYD, I. A. & FORRESTER, T. (1968) The release of adenosine triphosphate from frog skeletal musclein vitro.J. Physiol. 199, 115–35.

    Google Scholar 

  • BRADLEY, C., HENDERSON, W. B. & MILLER, D. J. (1980) Selectivity of Ca-Na antagonism in cardiac muscle.J. Physiol. 310, 78–9P.

    Google Scholar 

  • CLEMENS, M. G. & FORRESTER, T. (1981) Exacerbation of the calcium paradox with exogenous ATP in isolated rat heart.J. Physiol., Lond. 320, 121P.

    Google Scholar 

  • COCKCROFT, S. & GOMPERTS, B. D. (1979) Activation and inhibition of calcium-dependent histamine secretion by ATP ions applied to rat mast cells.J. Physiol. 296, 229–44.

    Google Scholar 

  • CORNELIUS, F. (1980) The regulation of tension in a chemically skinned molluscan smooth muscle.J. gen. Physiol. 75, 709–25.

    Google Scholar 

  • CRANK, J. (1967)The Mathematics of Diffusion, 1st edn. Oxford: Clarendon Press.

    Google Scholar 

  • DEITMAR, J. W. & ELLIS, D. (1980) Interactions between the regulation of the intracellular pH and sodium activity of sheep cardiac Purkinje fibres.J. Physiol. 304, 417–88.

    Google Scholar 

  • ELDER, H. Y., MILLER, D. J. & SMITH, G. L. (1981) Ultrastructural and contractile properties of Triton- and EGTA-treated rat heart.J. Physiol. 318, 33–4P.

    Google Scholar 

  • ENDO, M. & HNO, M. (1980) Specific perforation of muscle cell membranes preserved SR functions by saponin treatment.J. Musc. Res. Cell Motility 1, 89–100.

    Google Scholar 

  • FABIATO, A. & FABIATO, F. (1978) Effects of pH on the myofilaments and sarcoplasmic reticulum of skinned cells from cardiac and skeletal muscle.J. Physiol. 276, 233–55.

    Google Scholar 

  • FRY, C. H. & MILLER, D. J. (1984) The contribution of mitochondria to calcium metabolism and tension generation in cardiac muscle. InControl and Manipulation of Calcium Movement (edited by PARRATT, J.) pp. 87–106. New York: Raven.

    Google Scholar 

  • GETTES, L. S. & REUTER, H. (1974) Slow recovery from inactivation of inward currents in mammalian myocardial fibres.J. Physiol. 240, 703–24.

    Google Scholar 

  • GRINWALD, P. M. & NAYLER, W. G. (1981) Calcium entry in the calcium paradox.J. mol. cell. Cardiol. 13, 867–80.

    Google Scholar 

  • HARRISON, S. M. & MILLER, D. J. (1984) Mitochondrial contribution to relaxation demonstrated in skinned cardiac muscle.J. Physiol., Lond. (in press).

  • HEMPLING, H. G., STEWART, C. C. & GASIC, G. (1969) The effect of exogenous ATP on the electrolyte content of TA3 ascites tumor cells.J. Cell. Physiol. 73, 133–40.

    Google Scholar 

  • HIBBERT, M. G. & JEWELL, B. R. (1979) length-dependence of the sensitivity of the contractile system to calcium in rat ventricular muscle.J. Physiol., Lond. 290, 30P.

    Google Scholar 

  • HAAS, H. G., MEYER, R., EINWÄCHTER, H. M. & STOCKEM, W. (1983) Intercellular coupling in frog heart muscle. Electrophysiological and morphological aspects.Pflügers Arch. 399, 321–35.

    Google Scholar 

  • ISENBERG, G. & KLOCKNER, U. (1982) Calcium tolerant ventricular myocytes prepared by preincubation in a KB-medium.Pflügers Arch. 395, 6–18.

    Google Scholar 

  • JEWELL, B. R. & KENTISH, J. (1982) Can inotropic agents alter maximum force production of EGTA-treated trabeculae from rat ventricle?J. Physiol. 330, 81–2P.

    Google Scholar 

  • JOHANSSON, L. (1975) Some aspects of the constant ionic medium principle. Studies on Iron III Fluoride & Tris (propylenediamine) Cobalt III Iodide systems.Acta chem. scand. A29, 365–73.

    Google Scholar 

  • KENTISH, J. C. & JEWELL, B. R. (1984) Some characteristics of Ca2+-regulated force production in EGTA-treated muscles from rat heart.J. gen. Physiol. 84, 83–100.

    Google Scholar 

  • KERRICK, W. G. L. & BEST, P. M. (1974) Calcium ion release in mechanically disrupted heart cells.Science N.Y. 183, 435–7.

    Google Scholar 

  • KUSHMERICK, M. J. & PODOLSKY, R. J. (1969) Ionic mobility in muscle cells.Science N.Y. 166, 1297–8.

    Google Scholar 

  • LÉOTY, C. (1974) Membrane currents and activation of contraction in rat ventricular fibres.J. Physiol. 239, 237–49.

    Google Scholar 

  • LÜTTGAU, H. C. & SPIECKER, W. (1979) The effects of calcium deprivation upon mechanical and electrophysiological parameters in skeletal muscle fibres of the frog.J. Physiol. 296, 411–29.

    Google Scholar 

  • MARTELL, A. E. & SMITH, R. M. (1974)Critical Stability Constants. Vol. 1,Amino Acids. New York, London: Plenum Press.

    Google Scholar 

  • MCCLELLAN, G. B. & WINEGRAD, S. (1978) The regulation of the calcium sensitivity of the contractile system in mammalian cardiac muscle.J. gen. Physiol. 72, 737–64.

    Google Scholar 

  • MCCLELLAN, G. B. & WINEGRAD, S. (1980) Cyclic nucleotide regulation of the contractile proteins in mammalian heart muscle.J. gen. Physiol. 75, 283–95.

    Google Scholar 

  • McGUIGAN, J., CORAY, A., BOYETT, M., FRY, C., MILLER, D. J. & WEINGART, R. (1980) Na/Ca exchange in mammalian ventricular muscle. Prox. XXVIIIInt. Physiol. Sci. Congress (Budapest).

  • MILLER, D. J. (1975) Diffusion delays and the rate of contracture development in frog heart muscle.Pflügers Arch. 359, R23.

    Google Scholar 

  • MILLER, D. J. (1979a) Are cardiac muscle cells ‘skinned’ by EGTA or EDTA?Nature,277, 142–3.

    Google Scholar 

  • MILLER, D. J. (1979b) in ‘Matters arising’.Nature,280, 700–2.

    Google Scholar 

  • MILLER, D. J., ELDER, H. Y. & SMITH, G. L. (1985) Ultrastructural and X-ray microanalytical studies of EGTA- and detergent-treated heart muscle.J. Musc. Res. Cell Motility 6, 525–40.

    Google Scholar 

  • MILLER, D. J. & MOISESCU, D. G. (1976) The effects of very low external calcium and sodium concentrations on cardiac contractile strength and calcium-sodium antagonism.J. Physiol. 259, 283–308.

    Google Scholar 

  • MILLER, D. J. & MÖRCHEN, A. (1978) On the effects of divalent cations and ethylene glycol-bis-(betaaminooethyl) N,N,N',N'-tetraacetate on action potential duration in frog heart.J. gen. Physiol. 71, 47–67.

    Google Scholar 

  • MILLER, D. J., SINCLAIR, J., SMITH, A. D. & SMITH, G. L. (1982) Measurement of sarcomere length and automated exchange of bathing solutions applied to the study of intact and chemically ‘skinned’ cardiac muscle.J. Physiol., Lond. 320, 11P.

    Google Scholar 

  • MILLER, D. J. & SMITH, G. L. (1984a) EGTA purity and the buffering of Ca ions in physiological solutions.Am. J. Physiol. 246, C160–6.

    Google Scholar 

  • MILLER, D. J. & SMITH, G. L. (1984b) Diffusion equation modelling of the differences in the mechanical behaviour of EGTA- and Triton-treated rat heart.J. Physiol., Lond. 346, 74P.

    Google Scholar 

  • MOPE, L. G., MCCLELLAN, G. B. & WINEGRAD, S. (1980) Calcium sensitivity of the contractile system and phosphorylation of troponin in hyperpermeable cardiac cells.J. gen. Physiol. 75, 271–82.

    Google Scholar 

  • MORGAN, J. P., de FEO, T. T. & MORGAN, K. G. (1984) A chemical procedure for loading the calcium indicator aequorin into mammalian working myocardium.Pflügers Arch. 400, 338–40.

    Google Scholar 

  • MORGAN, J. P. & MORGAN, K. G. (1982) Vascular smooth muscle: The first recorded Ca2+ transients.Pflügers Arch. 395, 75–7.

    Google Scholar 

  • MORGAN, J. P. & MORGAN, K. G. (1984) Stimulus-specific patterns of intracellular calcium levels in smooth muscle of ferret portal vein.J. Physiol. 351, 155–68.

    Google Scholar 

  • PARKER, J. C., CASTRANOVA, V. & GOLDFINGER, J. M. (1977) Dog red blood cells: Na and K diffusion potentials with extracellular ATP.J. gen. Physiol. 69, 417–30.

    Google Scholar 

  • REQUENA, J., DIPOLO, R., BRINLEY, F. J. & MULLINGS, L. J. (1977) The control of ionized calcium in squid axons.J. gen. Physiol. 70, 329–53.

    Google Scholar 

  • REUBEN, J. P. & WOOD, D. S. (1979) In ‘Matters arising’.Nature 280, 700.

    Google Scholar 

  • ROUGIER, O., VASSORT, G., GARNIER, D., GARGOUIL, Y. M. & CORABOEUF, E. (1969) Existence and role of slow inward current during the frog atrial action potential.Pflügers Arch. 308, 91–110.

    Google Scholar 

  • SEEMAN, P. (1967) Transient holes in erythrocyte membranes during hypotonic hemolysis and stable holes in the membrane after lysis by saponin and lysolecithin.J. Cell Biol. 32, 55–7.

    Google Scholar 

  • SILINSKY, E. M. & HUBBARD, J. I. (1973) Release of ATP from rat motor nerve terminals.Nature 243, 404–5.

    Google Scholar 

  • SMITH, G. L. (1983)A functional and structural study of cardiac muscle subjected to membrane disruption techniques. Ph.D. Thesis, Glasgow University.

  • STEWAT, C. C., GASIC, G. & HEMPLING, H. G. (1969) Effect of exogenous ATP on the volume of TA3 ascites tumor cells.J. cell. comp. Physiol. 73, 125–32.

    Google Scholar 

  • SUTHERLAND, P. J., STEPHENSON, D. G. & WENDT, I. R. (1980) A novel method for introducing Ca2+-sensitive photoproteins into cardiac cells.Proc. Aust. physiol. pharmac. Soc. 11, 160P.

  • TODA, N. (1974) Automaticity induced by Ca2+ chelating agents in isolated rabbit left atria.Jpn. J. Pharmac. 24, 747–61.

    Google Scholar 

  • TRAMS, E. G. (1974) Evidence for ATP action on the cell surface.Nature 252, 480–2.

    Google Scholar 

  • VAUGHAN-JONES, R. D., EISNER, D. A. & LEDERER, W. J. (1983) Ca2+ ions can affect intracellular pH in mammalian cardiac muscle.Nature 301, 522–4.

    Google Scholar 

  • WEISBURG, A., MCCLELLAN, G., TUCKER, M., LIN, L. & WINEGRAD, S. (1983) Regulation of calcium sensitivity in perforated mammalian cardiac cells.J. gen. Physiol. 81, 195–211.

    Google Scholar 

  • WINEGRAD, S. (1971) Studies of cardiac muscle with a high permeability to calcium produced by treatment with ethylenediaminetetraacetic acid.J. gen. Physiol. 58, 71–93.

    Google Scholar 

  • WINEGRAD, S. (1973) Intracellular calcium binding and release in frog heart.J. gen. Physiol. 62, 693–706.

    Google Scholar 

  • WINEGRAD, S. (1979) In ‘Matters arising’.Nature 280, 700.

    Google Scholar 

Download references

Author information

Author notes

  1. Godfrey L. Smith

    Present address: Department of Physiology, University College London, Gower Street, WC1, London

Authors and Affiliations

  1. Institute of Physiology, University of Glasgow, G12 8QQ, Glasgow, U.K.

    David J. Miller & Godfrey L. Smith

Authors
  1. David J. Miller
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Godfrey L. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Miller, D.J., Smith, G.L. The contractile behaviour of EGTA- and detergent-treated heart muscle. J Muscle Res Cell Motil 6, 541–567 (1985). https://doi.org/10.1007/BF00711914

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00711914

Keywords

Search

Navigation