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Cell and Tissue Research

, Volume 216, Issue 2, pp 231–251 | Cite as

Oxygen requirements, morphology, cell coat and membrane permeability of calcium-tolerant myocytes from hearts of adult rats

  • Beatrice A. Wittenberg
  • Thomas F. Robinson
Article

Summary

The morphological, functional, and biochemical properties of freshly isolated heart muscle cells were examined. A reproducible method for the separation and purification of such cells isolated from adult rat heart was developed. It yields an average of 5×106 striated rectangular cells which retain normal morphology (range) 2.5 to 11×106 and 4×106 calcium-tolerant cells (range) 2.5 to 5.5×106 per heart. After purification, 85 to 95% of the cells retain normal morphology in solutions of calcium ion activity equal to 10μM, and 65 to 79% of the cells are rectangular in solutions of calcium ion activity equal to 1 mM.

Under the light microscope we were able to identify functionally intact individual cells that are calcium-tolerant and contract only in response to electrical stimulation, as well as dying myocytes that beat spontaneously. The examination of such cells under the electron microscope permitted us to address the question: What is the sequence of structural changes in a dying cell? The sarcomere lengths measured both in the living state and after preparation for electron microscopy are in the physiological range. In steady states of oxygen tension, respiration of the intact cells is undiminished from 50 torr to 2 torr. The oxygen tension for half maximal respiration is 0.15 torr. Therefore, the limitation of oxygen diffusion to the mitochondria of isolated heart muscle cells must be remarkably small.

Key words

Isolated cardiac myocytes Morphology Calcium-tolerance Oxygen requirement of respiration 

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References

  1. Anversa P, Loud AV, Giacomelli F, Wiener J (1978) Absolute morphometric study of myocardial hypertrophy in experimental hypertension. II. Ultrastructure of myocytes and interstitium. Lab Invest 38:597–608PubMedGoogle Scholar
  2. Arnold G, Lochner W (1965) Die Temperaturabhängigkeit des Sauerstoffverbrauches stillgestellter künstlich perfundierter Warmblüterherzen zwischen 34° and 4° C. Pflueger's Arch 284:169–175Google Scholar
  3. Berry MN, Friend DS, Scheuer J (1970) Morphology and metabolism of intact muscle cells isolated from adult rat heart. Circ Res XXVI: 679–687Google Scholar
  4. Bishop SP, Drummond JL (1979) Surface morphology and cell size measurement of isolated rat cardiac myocytes. J Mol Cell Cardiol 11:423–433Google Scholar
  5. Brady AJ, Tan ST, Ricchiuti NV (1979) Contractile force measured in unskinned isolated adult rat heart fibers. Nature 282:728–729Google Scholar
  6. Brown LM, Hill LM (1979) A new technique for rapid fixation of skeletal muscle. J Physiol (London) 289:8PGoogle Scholar
  7. Burns AH, Reddy WJ (1978) Amino acid stimulation of oxygen and substrate utilization by cardiac myocytes. Am J Physiol 235:E461–466Google Scholar
  8. Carlson EC, Grosso DS, Romero SA, Frangakis CJ, Byus CV, Bressler R (1978) Ultrastructural studies of metabolically active isolated adult rat heart myocytes. J Mol Cell Cardiol 10:449–459Google Scholar
  9. Clark MG, Gannon BJ, Bodkin N, Patten GS, Berry MN (1978) An improved procedure for the high-yield preparation of intact beating heart cells from the adult rat. Biochemical and Morphological Study. J Mol Cell Cardiol 10:1101–1121Google Scholar
  10. Cole RP, Sukanek PC, Wittenberg JB, Wittenberg BA (1980) Mitochondrial function in the presence of myoglobin. Submitted for publicationGoogle Scholar
  11. Fabiato A, Fabiato F (1975) Contractions induced by a calcium-triggered release of calcium from the sarcoplasmic reticulum of single skinned cardiac cells. J Physiol 249:469–495Google Scholar
  12. Fabiato A, Fabiato F (1979) Tension developed and intracellular free calcium concentration reached during the twitch of an isolated cardiac cell with closed sarcolemma. J Gen Physiol 74:6aGoogle Scholar
  13. Farmer BB, Harris RA, Jolly WW, Hathaway DR, Katzberg A, Watanabe AM, Whitlow AL, Besch HR Jr (1977) Isolation and characterization of adult rat heart cells. Arch Bioch Biophys 179:545–558Google Scholar
  14. Frangakis CJ, Bahl JJ, McDaniel H, Bressler R (1980) Tolerance to physiological calcium by isolated myocytes from the adult rat heart; an improved cellular preparation. Life Sciences 27:815–825Google Scholar
  15. Frank JS, Beydler S, Kreman M, Ron EE (1980) Structure of the freeze-fractured sarcolemma in the normal and anoxic rabbit myocardium. Circ Res 47:(1) 131–143Google Scholar
  16. Fry DM, Scales D, Inesi G (1979) The ultrastructure of membrane alterations of enzymatically dissociated cardiac myocytes. J Mol Cell Cardiol 11:1151–1163Google Scholar
  17. Gamble WJ, Conn PA, Kumar E, Plenge R, Monroe RG (1970) Myocardial oxygen consumption of blood-perfused, isolated, supported rat heart. Am J Physiol 219:604–612Google Scholar
  18. Gibbs CL (1978) Cardiac Energetics. Physiol Rev 58:174–254Google Scholar
  19. Glick MR, Burns AH, Reddy WJ (1974) Dispersion and isolation of beating cells from adult rat heart. Anal Biochem 61:32–42Google Scholar
  20. Harary I, Farley B (1963) In vitro studies on single beating rat heart cells. I Growth and organization. Exp Cell Res 29:451–465Google Scholar
  21. Haworth RA, Hunter DR, Berkoff HA (1980) The isolation of Ca2+-resistant myocytes from the adult rat. J Mol Cell Cardiol 12:715–723Google Scholar
  22. Irisawa H (1978) Comparative physiology of the cardiac pacemaker mechanism. Physiol Rev 58:461–498Google Scholar
  23. Isenberg G, Klöckner U (1980) Glycocalyx is not required for slow inward calcium current in isolated rat heart myocytes. Nature 284:358–360Google Scholar
  24. Jones DP, Mason HS (1978) Gradients of oxygen concentration in hepatocytes. J Biol Chem 253:4874–4880Google Scholar
  25. Katzberg AA, Farmer BB, Harris RA (1977) The predominance of binucleation in isolated rat heart myocytes. Am J Anat 149:489–500Google Scholar
  26. Krueger JW, Forletti D, Wittenberg BA (1980) Uniform sarcomere shortening behavior in isolated cardiac muscle cells. J Gen Physiol, in pressGoogle Scholar
  27. Mark GE, Strasser FF (1966) Pacemaker activity and mitosis in cultures of newborn rat heart ventricle cells. Exp Cell Res 44:217–233Google Scholar
  28. Moore EW, Ross JW (1965) NaCl and CaCl2 activity coefficients in mixed aqueous solutions. J Appl Physiol 20:1332–1336Google Scholar
  29. Moses RL, Kasten FH (1979) Ultrastructure of dissociated adult mammalian myocytes. J Mol Cell Cardiol 11:161–172Google Scholar
  30. Muir AR (1967) The effects of divalent cations on the ultrastructure of the perfused rat heart. J Anat 101:239–261Google Scholar
  31. Nag AC, Zak R (1979) Dissociation of adult mammalian heart into single cell suspension: an ultrastructural study. J Anat 129:541–559Google Scholar
  32. Nag AC, Fishman DA, Aumont MC, Zak R (1977) Studies of isolated adult rat heart cells: The surface morphology and the influence of extracellular calcium ion concentration on cellular viability. Tissue and Cell 9:419–436Google Scholar
  33. Neely JR, Liebermeister H, Battersby EJ, Morgan HE (1967) Effect of pressure development on oxygen consumption by isolated rat heart. Am J Physiol 212:804–814Google Scholar
  34. Page E, Upshaw-Early J (1977) Volume changes in sarcoplasmic reticulum of rat heart perfused with hypertonic solutions. Circ Res 40:355–366Google Scholar
  35. Page S (1974) Measurements of structural parameters in cardiac muscle. In: Ciba Foundation Symposium 24 (new series) Amsterdam: ElsevierGoogle Scholar
  36. Penparkgul S, Scheuer J (1969) Metabolic comparisons between hearts arrested by calcium deprivation or potassium excess. Am J Physiol 217:1405–1421Google Scholar
  37. Pepe FA (1971) Structure of the myosin filament of striated muscle. Prog Biophys Molec Biol 22:77–96Google Scholar
  38. Phillips HJ (1973) Dye exclusion tests for cell viability. In: Paul F, Kruse Jr, Patterson MK Jr (eds) Tissue Culture: Methods and Applications. Academic Press, New York, p 406–408Google Scholar
  39. Powell T, Steen EM, Twist VW, Woolf N (1978) Surface characteristics of cells isolated from adult rat myocardium. J Mol Cell Cardiol 10:287–292Google Scholar
  40. Powell T, Terrar DA, Twist VW (1980) Electrical properties of individual cells isolated from adult rat ventricular myocardium. J Physiol 302:131–153Google Scholar
  41. Robinson TF (1980) Lateral connections between heart muscle cells as revealed by conventional high voltage transmission electron microscopy. Cell Tissue Res 211:353–359Google Scholar
  42. Robinson TF, Winegrad S (1979) The measurement and dynamic implications of thin filament lengths in heart muscle. J Physiol 286:607–619Google Scholar
  43. Robinson TF, Aronson RS, Cohen-Gould L, Sorenson AL, Wittenberg BA, Sonnenblick EH (1980a) Heart muscle cells enzymatically isolated from ventricular wall: case studies of myofilament overlap configurations. Proc 38th Elec Micr Soc Amer 574–575Google Scholar
  44. Robinson TF, Hayward BS, Krueger JW, Sonnenblick EH, Wittenberg BA (1980b) Isolated heart myocytes: ultrastructural case study technique. Submitted for publicationGoogle Scholar
  45. Sato N, Hagihara B, Kamada T, Abe H (1976) A sensitive method for the quantitative estimation of cytochromes a and a3 in tissues. Anal Biochem 74:105–117Google Scholar
  46. Schuder S, Wittenberg JB, Haseltine B, Wittenberg BA (1979) Spectrophotometric determination of myoglobin in cardiac and skeletal muscle: Separation from hemoglobin by subunit-exchange chromatography. Anal Biochem 92:473–481Google Scholar
  47. Seglen PO (1976) Preparation of isolated rat liver cells. In: Prescott M (ed) Methods in Cell Biol XIII, 29–83. Academic Press, New YorkGoogle Scholar
  48. Simon W, Amman D, Oehme M, Murff WE (1978) Calcium-selective electrodes. Ann NY Acad Sci 307:52–69Google Scholar
  49. Spurr AR (1969) A low viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26:31–43PubMedGoogle Scholar
  50. Tamura M, Oshino N, Chance B, Silver IA (1978) Optical measurements of intracellular oxygen concentration of rat heart in vitro. Arch Bioch Biophys 191:8–22Google Scholar
  51. Venable JH, Coggeshall R (1965) A simplified lead citrate stain for use in electron microscopy. J Cell Biol 25:407–408CrossRefPubMedGoogle Scholar
  52. Wittenberg BA (1979) Myoglobin in isolated adult heart cells. In: Caughey W (ed) Biochem and Clin Aspects of oxygen. Acad Press, New York, p 35–51Google Scholar
  53. Wittenberg BA, Wittenberg JB, Krueger JW (1978) Myoglobin in oxygen economy of isolated cardiac cells. Fed Proc 37:1608Google Scholar
  54. Wittenberg JB (1970) Myoglobin-facilitated oxygen diffusion: role of myoglobin in oxygen entry into muscle. Physiol Rev 50:559–636Google Scholar

Copyright information

© Springer-Verlag 1981

Authors and Affiliations

  • Beatrice A. Wittenberg
    • 1
  • Thomas F. Robinson
    • 1
  1. 1.Departments of Physiology and MedicineAlbert Einstein College of MedicineBronxUSA

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