- Email: [email protected]
Cardiac Purkinje cells in culture
J Mol
Cell
Cardiol
15, 197-206
(1983)
Cardiac E. Canalel*,
Purkinje J. H. Campbell2
Cells
in Culture
and
G. R. Campbell1
l Department of Anatomy, University of Melbourne, Parkville, 3.052, and 2 Baker Medical Research Institute, Commercial Road, Prahran, 3181, Victoria, Australia (Received 13 October 1982, accepted in revisedform
24 November 1982)
E. CANALE, J. H. CAMPBELL AND G. R. CAMPBELL. Cardiac Purkinje Cells in Culture. Journal of Molecular and Cellular Cardiology (1983) 15, 197-206. Purkinje cells from false tendons of young rabbits, pigs and fetal lambs were dispersed by the action of collagenase and elastase and grown in culture for up to 14 days. Immunofluorescent staining with fluorescein-labelled antibodies to cardiac myosin and tropomyosin demonstrated cross-banding and/or a diffuse positive stain in Purkinje cells between 3 and 7 days in culture. Electron microscopy of cultured Purkinje cells at 3 days and 7 days revealed some disorganization of the myofilament system, in particular loss of Z-band material, as well as many thickened Z-bands, 120 nm to 240 nm in width. Gap junctions remained but desmosomes and fasciae adherentes were fewer in number. Organelles such as ribosomes, glycogen and mitochondria did not alter. Some Purkinje cells were spontaneously contractile in culture for up to seven days. Dominguez and Fozzard [7] propose that buckling of the Purkinje fibre and the production of sarcolemmal folds on the cell surface affect conduction of electrical impulses. We suggest that Purkinje cell contraction may play a major part in producing these geometric changes affecting conduction. KEY WORDS: Purkinie fibre: Culture of Purkinie: Ultrastructure; Phase contrast micro*scopy; Gap junctions. * ’
Introduction Physiological, pharmacological and biochemical studies on cultured cells have the advantage of both a controlled environment for experimentation and an extended viability for the tissue not available to other in vitro methods. Cell culture is one approach used widely in the study of the cell biology and embryology of the ventricular working myocardium, but it has not been applied to the specialized ‘conduction system of the heart. The present paper describes a method for the isolation and growth of cardiac Purkinje cells in culture. The significance of Purkinje fibre contractility in vivo is.also discussed in light of the finding that these fibres contract spontaneously in culture. Methods Cell Culture Hearts were obtained from freshly killed 2 to 6-week-old rabbits, 9 to 12-week-old pigs or fetal lambs 100 days’ gestation to newborn. * To whom
requests
0022-2828/83/030197+
for offprints 10 $03.00/O
should
Immunofluorescence;
Myoafilaments;
Left and right ventricles were opened and the false tendons dissected free in Hanks’ balanced salt solution, cut into Z-mm lengths and placed in serum-free Eagle’s Minimal Essential Medium with Hanks’ salts containing 3 mg/ml collagenase (Worthington CLS4196) at 37°C. After 30’to 45 min the solution was removed and replaced with freshly prepared 1 mg/ml elastase (Type III, N”E-0127 Sigmar) in serum-free medium for 1 h at 37”C, after which fresh collagenase (3 mg/ml) was added. When the pieces of false tendons were sufficiently dispersed (approximately 45 min:), the cell suspension was centrifuged at 900 r/min for 4 min. The pellet was then resuspended in medium with IO:/, foetal calf serum, preplated onto a plastic culture dish (Sterilin), and left at 37°C for 30 min, during which time many of the fibroblasts and smooth muscle cells attached to the surface of the dish. The Purkinje-cell enriched suspension was then either re-plated into collagen-coated culture dishes or injected into Rose chambers.
be sent. 0
1983 Academic
Press Inc.
(London)
Limited
198
E. Canale
et al.
Electron Microscopy Cultures taken for electron microscopy were fixed in 5% glutaraldehyde in 0.1 M phosphate buffer for 15 min, post-fixed in osmium tetroxide, dehydrated in alcohol and embedded in epon-araldite. Thin sections were stained with uranyl acetate and Reynold’s lead citrate, viewed and photographed with a Siemens Elmiskop 102 or Philips 400T electron microscope.
mained viable for at least 14 days in culture (longest time studied). Over this time the cells did not alter their appearance with phasecontrast microscopy, and were always clearly distinguishable from the fibroblasts which eventually overgrew the culture. Mitosis of Purkinje cells was not observed with phasecontrast microscopy, but in fixed cultures stained with labelled antibodies, mitotic figures were sometimes seen.
ImmunoJuorescence Cultures for immunofluorescence were grown on glass coverslips, rinsed in phosphate buffered saline (PBS), fixed in 2q/, paraformaldehyde for 5 min then rinsed again in PBS. Cell membranes were disrupted by washing in acetone for 5 min at -15°C. Using the indirect technique, cells were labelled with antibodies to either chicken cardiac myosin or tropomyosin prepared in the rabbit, then with fluorescein-conjugated rabbit IgG [lo]. Antibodies to chicken cardiac myosin stain A-bands of cardiac muscle but do not stain smooth muscle, fibroblasts, endocardium or endothelial cells [9]. Antibodies to tropomyosin stain Ibands of cardiac muscle [5].
ImmunoJuorescence Purkinje cells in culture stained strongly with fluorescein-conjugated antibodies to either cardiac myosin or tropomyosin. After 3 days .in culture the cells exhibited a cross-banding pattern due to the sarcomeric arrangement of myofibrils as well as fibres without banding and a more diffuse staining (Figure 2). By seven days, a greater number of cells showed more diffuse staining and non-striated fibrils than sarcomeric cross-banding. Purkinje cells in clumps maintained a close association with each other and were often connected by long cytoplasmic extensions (Figure 2).
Results
Phase Con$zst Microscopy Purkinje cells from the rabbit, pig and lamb behaved similarly in culture. Immediately upon culture, while the cells were still in suspension, spontaneously contracting Purkinje cells -were visible by phase-contrast microscopy as single cells and in groups of varying sizes. Their rate of contraction was approximately 15 to 30/min. Contractions in some cells were observed after seven days in culture although most ceased contracting after three days. By the third or fourth day the Purkinje cells had flattened onto the culture substrate and were surrounded by fibroblasts (Figure 1). Purkinje cells were conspicuously larger than the fibroblasts and some had striations in their cytoplasm (Figure 1a). Many were binucleate, each nucleus being spherical and having a prominent nucleolus. All cells re-
Electron Microscopy By day 3 a whole spectrum of morphologies were present within each clump of Purkinje cells, with those cells at the periphery most altered from in vivo. After 7 days all Purkinje fibres observed with the transmission electron microscope resembled those at the periphery of clumps with the following morphological changes. The most obvious change was disorganization of the myofilament system, with Z-band material as well as actin and myosin filaments dispersed in the cytoplasm. These cells contained large numbers of ribosomes, mitochondria and sometimes a few lipid droplets (Figure 3a). Sub-sarcolemmal dyads had a normal appearance although they appeared to be fewer in number. Gap junctions were present (Figure 3b), but there were few desmosomes, and fasciae adherentes appeared as an electron dense accumulation along the membrane. Normal sized myofibrils were observed lacking Z-bands, or with thickened Z-bands, 120 nm to 240 nm in width (Figures 4, 5a, 5c) and were probably responsible for the
1FIGURE 1. Phase dense micrographs stri ations (S) and surrounding fibroblasts (F) . Bar length = 20 pm.
of Purkinje cells after four days in culture. (a) Pig Purkinje cells (P) with (F). (b) Lamb Purkinje cells (P) with no striations visible. Fibrol bhasts
F ‘IGURE 2. Rabbit Purkinje foul : days in culture. (a) Many but most of the staining does leng :th = 20 I”.
cells stained with fluorescein-labelled striated fibrils are present (arrowhead). not show striations. Some cells form
after antibodies to chick cardiac myosin (b) A few striations can be seen (a;rrou Ihead) long cytoplasmic extensions (umw) . Bar
FIGURE 3. Purkinje cells after 3 days in culture. (a) Myosin Z-band material (circled) can be seen in the cytoplasm. Bar strating a gap junction (gj) and myofilaments (my). Bar length
filaments (*) may be in bundles length = 0.1 pm. (b) Boxed area = 50 nm.
or disorganized of 3(a) demon-
F ‘IGURE MY ofibrils
4. Purkinje cell after 7 days in culture. Myofibrillar have a lack of Z-band material (arrowed) or thickened
disorganization Z-bands (circled).
is no greater thail Lipid (L). Bar length
at 3 days. = 0.1 Wm.
‘IGURE 5. Purkinje cell after 7 days in culture. ribosomes (r) and Z-band (<) are all present. merits (m). Z-band material (<), ribosomes (r). of 120 to 240 nm. Mitochondria hav ,ing a thickness
(a) Some myofibrils (m) are without Z-bands. Actin filaments (b) Some areas have many actin filaments (u) but few myosin than usually encountered in uioo, (c) Z-bands (<) were thicker (mi). Bar length = 50 nm.
204
E. Canale
cross-banding pattern observed with immunofluorescent staining. No Z-bands of normal thickness were observed in culture. The areas of cytoplasm with haphazardly dispersed myosin and actin filaments [Figures 3(a), 5(b)], would account for the diffuse type of staining observed with immunofluorescence. Discussion
This is the first report of a technique for the culture of cardiac Purkinje cells. With further development it could provide a useful tool for the study of the biology of these cells. For example, the value of culture for the study of cardiac Purkinje cells lies in the possibility of altering in vitro conditions to mimic the changing physiology and pathology occurring in vivo. Well controlled experiments on the effects of changing single parameters (e.g.. hypoxia) could be studied over many days, and the effects of new drugs studied without neural or hormone-mediated effects superimposed on direct actions of the drugs on Purkinje cells. This kind of information would allow better evaluation of the therapeutic value of drugs, especially their potential as antiarrhythmic agents. The main difficulty encountered in culturing cardiac Purkinje cells was removal of the dense connective tissue sheath surrounding the fibres. This was achieved by use of the enzymes collagenase and elastase which are routinely used in our laboratory to dissociate aortic smooth muscle cells from their extensive connective tissue matrix [4]. Contamination by working myocardium was avoided by culturing only from false tendons in which Purkinje cells are usually the only myocardial cell type found [2]. Our technique at present maintains viable Purkinje cells for at least two weeks, but with time in culture Purkinje cells appear to undergo a form of dedifferentiation. Phenotypic modulation of isolated smooth muscle cells in culture is well known [S], while working myocardial cells remain contractile for many nionths in culture [13]. Morphological changes were observed in cultured Purkinje celIs, both by immuno-fluorescent labelling of cardiac myosin and tropomyosin, and in transmission electron microscopy. Immuno-
et al. fluorescent labelling of cardiac myosin or tropomyosin in working myocardium reveals a cross-banding pattern due to the sarcomerit organization of myofibrils, irrespective of whether the cells are one day or one month in culture [13]. However, after 3 days in culture Purkinje cells reveal a diffuse pattern of fibrils as well as sarcomeric cross-banding after myosin staining. This correlates exactly with electron microscopy of the cells which shows disarranged myosin and actin filaments as well as the disappearance of much Z-band material. A similar disorganization has recently been reported in isolated nodal cells which have been kept intact for 2 days in salt solution [14]. The changes observed in the myofilament system of Purkinje cells may be due to some factor(s) present in uivo that have not been reproduced in the culture environment. Factors to be considered for such a role include mechanical stress, neural factors and the influence of working myocardium as well as other Purkinje fibres. Evidence has already been put forward that the directions of mechanical stress are responsible for the alignment of myofibrils in Purkinje cells [29], but it is not known to what degree changes in the magnitude of stress influences myofibrillogenesis. A significant result in this study is the observation that Purkinje cells contract spontaneously in culture. Since the function of the Purkinje cells is conduction rather than adding to the contractile force of the heart, the exact function of the myofibrils in these cells has been uncertain. Early electron microscopists detected imperfections and a level of disorganization in myofibrils of Purkinje cells not seen in working myocardium [3, 2.51. Muir [15] called these “Purkinje fibrils” to distinguish them from the normal myofibrils seen in Purkinje cells and working myocardium. Caesar et al. [3] emphasized their embryonic appearance and described a second filament system of leptofibrils in sheep Purkinje fibres. Thickened Z-bands is one distinguishing feature of Purkinje cell myofibrils in viuo [26] as well as in vitro. In vivo, the best organized and well developed myofibrils occur peripherally in the cell and particularly where two Purkinje cells are joined laterally. These areas corres-
Cardiac
Purkinje
pond to the same regions where the incidence of sub-sarcolemmal dyads is highest [16]. The dyads are thought to be involved in excitation-contraction coupling, being the sites where the sarcoplasmic reticulum receives the signal from the depolarized sarcolemma to release calcium which initiates myofibrillar contraction. The observation that sub-sarcolemmal dyads are more common where myofibrils are most abundant further implicates them in this role. In a number of publications [3, 8, 15, 17, 18, 21, 22, 24-301 and particularly in two major reviews [23, 271, attention has centred on the differences between Purkinje and working myocardium filament systems and the absence of T-tubules in Purkinje cells. The absence of a T-tubular system has been used as an argument against Purkinje cell contraction. However, the working myocardium of birds is extremely efficient in contraction and is without T-tubules [I, 201. The absence of a T-tubular system may be related to the small size of avian working myocardial cells if one of the functions of T-tubules is to ensure simultaneous contraction of all myofibrils in large muscle cells and minimize the delay between excitation and the initiation of contraction. Therefore, in the very large Purkinje cells a T-tubular system may not be necessary if the myofibrils to be activated are located near the sarcolemma. The demonstration of spontaneous contraction by Purkinje cells raises the question of the possible function of Purkinje cell contraction in vivo. It is highly unlikely that the Purkinje system could add appreciable force
Cells in Culture
2105
to ventricular contraction because of tlhe cable-like geometry of the system and the low level of development of the contractile apparatus in comparison to the working myocardium. Nevertheless, based on tlhe observations of this paper, it would Ibe incorrect to regard myofibrils in Purkinje cells as “vestigial organelles” [17], or as being only cytoskeletal or elastic in nature [27]. Recently, contractions of sheep cardi,ac Purkinje fibres kept in Tyrode’s solution have been reported [II, 121. Dominguez and Fozzard [7], and Sanders et al. [19] have demonstrated that Purkinje fibre geometry alters with stretch, affecting conduction in the fibre. Due to the effects of buckling of tlhe fibre and folding of the Purkinje sarcolemma, conduction time across a false tendon remains almost constant at different stretch lengths of the false tendon. This ensures constant myocardial activation times at different amounts of diastolic ventricular filling. Purkinje cell contraction is probably responsible for the buckling and folding described by Dominguez and Fozzard [7]. This suggestion is an appealing one because it integrates a contractile function of the Purkinje cell with jits known major function of conduction. Acknowledgements We wish to thank Professor U. GroschelStewart, Zoologisches Institut, Darmstaclt, West Germany, for her generous gift of antibodies to tropomyosin and myosin. This work was supported by the National Heart Foundation of Australia.
References 1. 2.
3. 4.
5.
6.
AKESTER, A. R. Intercalated discs, nexuses, sarcoplasmic reticulum and transitional cells in the heart of the adult domestic fowl (Callus Gallus Domesticus). J Anat 133, 161-179 (1981). BENCOSME, S. A., TRILLO, A., ALANIS, J., BENITEZ, D. Correlative ultrastructural and electrophysiological study of the Purkinje system of the heart. J Electrocardiol2,27-28 ( 1969). CAESAR, R., EDWARDS, G. A., RUSKA, H. Electron microscopy of the impulse conducting system of the sheep heart. 2 Zellforsch Mikrosk Anat 48, 698-719 (1958). CHAMLEY, J. H., CAMPBELL, G. R., MCCONNELL, J. D., GRBSCHEL-STEWART, U. Comparison of vascular smooth muscle cells from adult human, monkey and rabbit in primary culture and in subculture. Cell Tissue Res 177,503-522 (1977). CHAMLEY-CAMPBELL, J. ‘H., CAMPBELI,, G. R., GRGSCHEL-STEWART, U., BURNSTOCK, G. FITC-labelled antibody staining of tropomyosin-containing fibrils in smooth, cardiac and skeletal muscle cells, pre-fusion myoblasts, fibroblasts, endothelial cells and 3T3 cells in culture. Cell Tissue Res 183, 153-166 (1977). CHAMLEY-CAMPBELL, J. H., CAMPBELL, G. R., Ross, R. The smooth muscle cell in culture. Physiol Rev 59, 1-61 (1979).
206 7.
E. Canale
G., FOZZARD, H. A. Effect of stretch on conduction velocity and cable properties of cardiac fibres. Am J Physio1237, Cl 19X124 (1979). ERIKSSON, A., THORNELL, L.-E. Intermediate (skeletin) filaments in heart Purkinje fibres. J Cell Biol 80, 231-247 (1979). GR~SCHEL-STEWART, U., CHAMLEY, J. H., MCCONNELL, J. D., BURNSTOCK, G. Comparison of the reaction of cultured smooth and cardiac muscle cells and fibroblasts to specific antibodies to myosin. Histochemistry 43, 215-224 (1975). GR~SCHEL-STEWART, U., SCHREIBER, J., MAHLMEISTER, C., WEBER, K. Production of specific antibodies to contractile proteins, and their use in immunofluorescence microscopy. I. Antibodies to smooth and striated chicken muscle myosins. Histochemie 46,229-236 (1976). LIPSIUS, S. L., FOZZARD, H. A., GIBBONS, W. R. Voltage and time dependence of restitution in heart. Am J Physio1243 (Heart Circ Physiol 12) H68-H76 (1982). Lrps~us, S. L., GIBBONS, W. R. Membrane currents, contractions and aftercontractions in cardiac Purkinje fibres. Am J Physiol 243 (Heart Circ Physiol 12) H77-H86 (1982). MARK, G. E., CHAMLEY, J. H., BURNSTOCK, G. Interactions between autonomic nerves and smooth and cardiac muscle cells in tissue culture. Dev Biol 32, 194-200 (1973). MASSON-P&ET, M., JONGSMA, H. J., BLEEKER, W. K., TSJERNINA, L., VAN GINNEKEN, A. C. G., TREI~L, B. W., BOUMAN, L. N. Intact isolated sinus node cells from the adult rabbit heart. J Moi Cell Cardiol 14, 295-299 ( 1982). MUIR, A. R. Observations on the fine structure of the Purkinje fibres in the ventricles of the sheeps heart. J Anat 91, 251-258 (1957). N~AEz-Du&, H. Electron microscopic study of the sarcolemma of Purkinje cells of the goat heart. Acta Anat 109,19-24 (1981). OLIPHANT, L. W., LOEWEN, R. D. Filament systems in Purkinje cells of sheep heart: Possible alteration of myofibrillogenesis. J Mol Cell Cardiol 8, 679-688 (1976). SAITO, K., TAMURA, Y., SAITO, M., MATSUMURA, K., NIKI, T., MORI, H. Comparison of the subunit compositions and ATPase activities of myosin in the myocardium and conduction system. J Mol Cell Cardiol 13, 331-322 (1981). SANDERS, R., MYERBURG, R. J,, GELBAND, H., BASSETT, A. L. Dissimilar length-tension relations of canine ventricular muscle and false tendon: Electrophysiologic alterations accompanying deformation. J Mel .Cell Cardiol l&209-219 (1979). SCOTT, T. M. The ultrastructure of ordinary and Purkinje cells of the fowl heart. J Anat 110, 259-273 (1971). SOMMER, J. R., JOHNSON, E. A. Cardiac muscle:’ A comparative study of Purkinje fibrcs and ventricular fibres. J Cell Biol 36, 497-526 (1968). SOMMER, J. R., JOHNSON, E. A. Purkinje fibres of the heart examined with the peroxidase reaction. J Cell Biol 37, 570-574 (1968). SOMMER, J. R., JOHNSON, E. A. Ultrastructure of cardiac muscle. In: Handbook of Physiology, vol. 1, section 2, “The Heart”, Eds. Berne, R. M., Sperelakis, N. American Physiological Society, pp. 113-186 (1979). THORNELL, L.-E. Myofilament-polyribosome complexes in the conducting system of hearts from cow, rabbit and cat. J Ultrastruct Res 41, 579-596 (1972). THORNELL, L.-E. Evidence of an imbalance in synthesis and degradation of myofibrillar proteins in rabbit Purkinje fibrcs. J Ultrastruct Rcs 44, 85-95 (1973). THORNELL, L.-E. Ultrastructural variations of Z-bands in cow Purkinje fibres. J Mol Cell Cardiol5,409-417 (1973). THORNELL, L.-E., ERIKSSON, A. Filament systems in the Purkinje fibres of the heart. Am J Physiol 241, H291-H305 (1981). THORNELL, L.-E., ERIKSSON, A., STIGBRAND, T., SJOSTROM, M. Structural proteins in cow Purkinje and ordinary ventricular fibres-a marked difference. J Mol Cell Cardiol 10,605-616 (1978). THORNELL, L.-E., SJOSTROM, M., ANDERSON, K.-E. The relationship between mechanical stress and myofibrillar organization in heart Purkinje fibres. J Mol Cell Cardio18, 689-695 (1976). VI~ACH, Sz., CHALLICE, C. E. Variations in filamentous and fibrillar organization, and associated sarcolemmal structures in cells of the normal mammalian heart. J Ultrastruct Res 28,321-334 (1969). DOMINGUEZ,
Purkinje
8. 9.
10.
11. 12. 13. 14.
15. 16. 17. 18.
19.
20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
et al.
Cell
Cardiol
15, 197-206
(1983)
Cardiac E. Canalel*,
Purkinje J. H. Campbell2
Cells
in Culture
and
G. R. Campbell1
l Department of Anatomy, University of Melbourne, Parkville, 3.052, and 2 Baker Medical Research Institute, Commercial Road, Prahran, 3181, Victoria, Australia (Received 13 October 1982, accepted in revisedform
24 November 1982)
E. CANALE, J. H. CAMPBELL AND G. R. CAMPBELL. Cardiac Purkinje Cells in Culture. Journal of Molecular and Cellular Cardiology (1983) 15, 197-206. Purkinje cells from false tendons of young rabbits, pigs and fetal lambs were dispersed by the action of collagenase and elastase and grown in culture for up to 14 days. Immunofluorescent staining with fluorescein-labelled antibodies to cardiac myosin and tropomyosin demonstrated cross-banding and/or a diffuse positive stain in Purkinje cells between 3 and 7 days in culture. Electron microscopy of cultured Purkinje cells at 3 days and 7 days revealed some disorganization of the myofilament system, in particular loss of Z-band material, as well as many thickened Z-bands, 120 nm to 240 nm in width. Gap junctions remained but desmosomes and fasciae adherentes were fewer in number. Organelles such as ribosomes, glycogen and mitochondria did not alter. Some Purkinje cells were spontaneously contractile in culture for up to seven days. Dominguez and Fozzard [7] propose that buckling of the Purkinje fibre and the production of sarcolemmal folds on the cell surface affect conduction of electrical impulses. We suggest that Purkinje cell contraction may play a major part in producing these geometric changes affecting conduction. KEY WORDS: Purkinie fibre: Culture of Purkinie: Ultrastructure; Phase contrast micro*scopy; Gap junctions. * ’
Introduction Physiological, pharmacological and biochemical studies on cultured cells have the advantage of both a controlled environment for experimentation and an extended viability for the tissue not available to other in vitro methods. Cell culture is one approach used widely in the study of the cell biology and embryology of the ventricular working myocardium, but it has not been applied to the specialized ‘conduction system of the heart. The present paper describes a method for the isolation and growth of cardiac Purkinje cells in culture. The significance of Purkinje fibre contractility in vivo is.also discussed in light of the finding that these fibres contract spontaneously in culture. Methods Cell Culture Hearts were obtained from freshly killed 2 to 6-week-old rabbits, 9 to 12-week-old pigs or fetal lambs 100 days’ gestation to newborn. * To whom
requests
0022-2828/83/030197+
for offprints 10 $03.00/O
should
Immunofluorescence;
Myoafilaments;
Left and right ventricles were opened and the false tendons dissected free in Hanks’ balanced salt solution, cut into Z-mm lengths and placed in serum-free Eagle’s Minimal Essential Medium with Hanks’ salts containing 3 mg/ml collagenase (Worthington CLS4196) at 37°C. After 30’to 45 min the solution was removed and replaced with freshly prepared 1 mg/ml elastase (Type III, N”E-0127 Sigmar) in serum-free medium for 1 h at 37”C, after which fresh collagenase (3 mg/ml) was added. When the pieces of false tendons were sufficiently dispersed (approximately 45 min:), the cell suspension was centrifuged at 900 r/min for 4 min. The pellet was then resuspended in medium with IO:/, foetal calf serum, preplated onto a plastic culture dish (Sterilin), and left at 37°C for 30 min, during which time many of the fibroblasts and smooth muscle cells attached to the surface of the dish. The Purkinje-cell enriched suspension was then either re-plated into collagen-coated culture dishes or injected into Rose chambers.
be sent. 0
1983 Academic
Press Inc.
(London)
Limited
198
E. Canale
et al.
Electron Microscopy Cultures taken for electron microscopy were fixed in 5% glutaraldehyde in 0.1 M phosphate buffer for 15 min, post-fixed in osmium tetroxide, dehydrated in alcohol and embedded in epon-araldite. Thin sections were stained with uranyl acetate and Reynold’s lead citrate, viewed and photographed with a Siemens Elmiskop 102 or Philips 400T electron microscope.
mained viable for at least 14 days in culture (longest time studied). Over this time the cells did not alter their appearance with phasecontrast microscopy, and were always clearly distinguishable from the fibroblasts which eventually overgrew the culture. Mitosis of Purkinje cells was not observed with phasecontrast microscopy, but in fixed cultures stained with labelled antibodies, mitotic figures were sometimes seen.
ImmunoJuorescence Cultures for immunofluorescence were grown on glass coverslips, rinsed in phosphate buffered saline (PBS), fixed in 2q/, paraformaldehyde for 5 min then rinsed again in PBS. Cell membranes were disrupted by washing in acetone for 5 min at -15°C. Using the indirect technique, cells were labelled with antibodies to either chicken cardiac myosin or tropomyosin prepared in the rabbit, then with fluorescein-conjugated rabbit IgG [lo]. Antibodies to chicken cardiac myosin stain A-bands of cardiac muscle but do not stain smooth muscle, fibroblasts, endocardium or endothelial cells [9]. Antibodies to tropomyosin stain Ibands of cardiac muscle [5].
ImmunoJuorescence Purkinje cells in culture stained strongly with fluorescein-conjugated antibodies to either cardiac myosin or tropomyosin. After 3 days .in culture the cells exhibited a cross-banding pattern due to the sarcomeric arrangement of myofibrils as well as fibres without banding and a more diffuse staining (Figure 2). By seven days, a greater number of cells showed more diffuse staining and non-striated fibrils than sarcomeric cross-banding. Purkinje cells in clumps maintained a close association with each other and were often connected by long cytoplasmic extensions (Figure 2).
Results
Phase Con$zst Microscopy Purkinje cells from the rabbit, pig and lamb behaved similarly in culture. Immediately upon culture, while the cells were still in suspension, spontaneously contracting Purkinje cells -were visible by phase-contrast microscopy as single cells and in groups of varying sizes. Their rate of contraction was approximately 15 to 30/min. Contractions in some cells were observed after seven days in culture although most ceased contracting after three days. By the third or fourth day the Purkinje cells had flattened onto the culture substrate and were surrounded by fibroblasts (Figure 1). Purkinje cells were conspicuously larger than the fibroblasts and some had striations in their cytoplasm (Figure 1a). Many were binucleate, each nucleus being spherical and having a prominent nucleolus. All cells re-
Electron Microscopy By day 3 a whole spectrum of morphologies were present within each clump of Purkinje cells, with those cells at the periphery most altered from in vivo. After 7 days all Purkinje fibres observed with the transmission electron microscope resembled those at the periphery of clumps with the following morphological changes. The most obvious change was disorganization of the myofilament system, with Z-band material as well as actin and myosin filaments dispersed in the cytoplasm. These cells contained large numbers of ribosomes, mitochondria and sometimes a few lipid droplets (Figure 3a). Sub-sarcolemmal dyads had a normal appearance although they appeared to be fewer in number. Gap junctions were present (Figure 3b), but there were few desmosomes, and fasciae adherentes appeared as an electron dense accumulation along the membrane. Normal sized myofibrils were observed lacking Z-bands, or with thickened Z-bands, 120 nm to 240 nm in width (Figures 4, 5a, 5c) and were probably responsible for the
1FIGURE 1. Phase dense micrographs stri ations (S) and surrounding fibroblasts (F) . Bar length = 20 pm.
of Purkinje cells after four days in culture. (a) Pig Purkinje cells (P) with (F). (b) Lamb Purkinje cells (P) with no striations visible. Fibrol bhasts
F ‘IGURE 2. Rabbit Purkinje foul : days in culture. (a) Many but most of the staining does leng :th = 20 I”.
cells stained with fluorescein-labelled striated fibrils are present (arrowhead). not show striations. Some cells form
after antibodies to chick cardiac myosin (b) A few striations can be seen (a;rrou Ihead) long cytoplasmic extensions (umw) . Bar
FIGURE 3. Purkinje cells after 3 days in culture. (a) Myosin Z-band material (circled) can be seen in the cytoplasm. Bar strating a gap junction (gj) and myofilaments (my). Bar length
filaments (*) may be in bundles length = 0.1 pm. (b) Boxed area = 50 nm.
or disorganized of 3(a) demon-
F ‘IGURE MY ofibrils
4. Purkinje cell after 7 days in culture. Myofibrillar have a lack of Z-band material (arrowed) or thickened
disorganization Z-bands (circled).
is no greater thail Lipid (L). Bar length
at 3 days. = 0.1 Wm.
‘IGURE 5. Purkinje cell after 7 days in culture. ribosomes (r) and Z-band (<) are all present. merits (m). Z-band material (<), ribosomes (r). of 120 to 240 nm. Mitochondria hav ,ing a thickness
(a) Some myofibrils (m) are without Z-bands. Actin filaments (b) Some areas have many actin filaments (u) but few myosin than usually encountered in uioo, (c) Z-bands (<) were thicker (mi). Bar length = 50 nm.
204
E. Canale
cross-banding pattern observed with immunofluorescent staining. No Z-bands of normal thickness were observed in culture. The areas of cytoplasm with haphazardly dispersed myosin and actin filaments [Figures 3(a), 5(b)], would account for the diffuse type of staining observed with immunofluorescence. Discussion
This is the first report of a technique for the culture of cardiac Purkinje cells. With further development it could provide a useful tool for the study of the biology of these cells. For example, the value of culture for the study of cardiac Purkinje cells lies in the possibility of altering in vitro conditions to mimic the changing physiology and pathology occurring in vivo. Well controlled experiments on the effects of changing single parameters (e.g.. hypoxia) could be studied over many days, and the effects of new drugs studied without neural or hormone-mediated effects superimposed on direct actions of the drugs on Purkinje cells. This kind of information would allow better evaluation of the therapeutic value of drugs, especially their potential as antiarrhythmic agents. The main difficulty encountered in culturing cardiac Purkinje cells was removal of the dense connective tissue sheath surrounding the fibres. This was achieved by use of the enzymes collagenase and elastase which are routinely used in our laboratory to dissociate aortic smooth muscle cells from their extensive connective tissue matrix [4]. Contamination by working myocardium was avoided by culturing only from false tendons in which Purkinje cells are usually the only myocardial cell type found [2]. Our technique at present maintains viable Purkinje cells for at least two weeks, but with time in culture Purkinje cells appear to undergo a form of dedifferentiation. Phenotypic modulation of isolated smooth muscle cells in culture is well known [S], while working myocardial cells remain contractile for many nionths in culture [13]. Morphological changes were observed in cultured Purkinje celIs, both by immuno-fluorescent labelling of cardiac myosin and tropomyosin, and in transmission electron microscopy. Immuno-
et al. fluorescent labelling of cardiac myosin or tropomyosin in working myocardium reveals a cross-banding pattern due to the sarcomerit organization of myofibrils, irrespective of whether the cells are one day or one month in culture [13]. However, after 3 days in culture Purkinje cells reveal a diffuse pattern of fibrils as well as sarcomeric cross-banding after myosin staining. This correlates exactly with electron microscopy of the cells which shows disarranged myosin and actin filaments as well as the disappearance of much Z-band material. A similar disorganization has recently been reported in isolated nodal cells which have been kept intact for 2 days in salt solution [14]. The changes observed in the myofilament system of Purkinje cells may be due to some factor(s) present in uivo that have not been reproduced in the culture environment. Factors to be considered for such a role include mechanical stress, neural factors and the influence of working myocardium as well as other Purkinje fibres. Evidence has already been put forward that the directions of mechanical stress are responsible for the alignment of myofibrils in Purkinje cells [29], but it is not known to what degree changes in the magnitude of stress influences myofibrillogenesis. A significant result in this study is the observation that Purkinje cells contract spontaneously in culture. Since the function of the Purkinje cells is conduction rather than adding to the contractile force of the heart, the exact function of the myofibrils in these cells has been uncertain. Early electron microscopists detected imperfections and a level of disorganization in myofibrils of Purkinje cells not seen in working myocardium [3, 2.51. Muir [15] called these “Purkinje fibrils” to distinguish them from the normal myofibrils seen in Purkinje cells and working myocardium. Caesar et al. [3] emphasized their embryonic appearance and described a second filament system of leptofibrils in sheep Purkinje fibres. Thickened Z-bands is one distinguishing feature of Purkinje cell myofibrils in viuo [26] as well as in vitro. In vivo, the best organized and well developed myofibrils occur peripherally in the cell and particularly where two Purkinje cells are joined laterally. These areas corres-
Cardiac
Purkinje
pond to the same regions where the incidence of sub-sarcolemmal dyads is highest [16]. The dyads are thought to be involved in excitation-contraction coupling, being the sites where the sarcoplasmic reticulum receives the signal from the depolarized sarcolemma to release calcium which initiates myofibrillar contraction. The observation that sub-sarcolemmal dyads are more common where myofibrils are most abundant further implicates them in this role. In a number of publications [3, 8, 15, 17, 18, 21, 22, 24-301 and particularly in two major reviews [23, 271, attention has centred on the differences between Purkinje and working myocardium filament systems and the absence of T-tubules in Purkinje cells. The absence of a T-tubular system has been used as an argument against Purkinje cell contraction. However, the working myocardium of birds is extremely efficient in contraction and is without T-tubules [I, 201. The absence of a T-tubular system may be related to the small size of avian working myocardial cells if one of the functions of T-tubules is to ensure simultaneous contraction of all myofibrils in large muscle cells and minimize the delay between excitation and the initiation of contraction. Therefore, in the very large Purkinje cells a T-tubular system may not be necessary if the myofibrils to be activated are located near the sarcolemma. The demonstration of spontaneous contraction by Purkinje cells raises the question of the possible function of Purkinje cell contraction in vivo. It is highly unlikely that the Purkinje system could add appreciable force
Cells in Culture
2105
to ventricular contraction because of tlhe cable-like geometry of the system and the low level of development of the contractile apparatus in comparison to the working myocardium. Nevertheless, based on tlhe observations of this paper, it would Ibe incorrect to regard myofibrils in Purkinje cells as “vestigial organelles” [17], or as being only cytoskeletal or elastic in nature [27]. Recently, contractions of sheep cardi,ac Purkinje fibres kept in Tyrode’s solution have been reported [II, 121. Dominguez and Fozzard [7], and Sanders et al. [19] have demonstrated that Purkinje fibre geometry alters with stretch, affecting conduction in the fibre. Due to the effects of buckling of tlhe fibre and folding of the Purkinje sarcolemma, conduction time across a false tendon remains almost constant at different stretch lengths of the false tendon. This ensures constant myocardial activation times at different amounts of diastolic ventricular filling. Purkinje cell contraction is probably responsible for the buckling and folding described by Dominguez and Fozzard [7]. This suggestion is an appealing one because it integrates a contractile function of the Purkinje cell with jits known major function of conduction. Acknowledgements We wish to thank Professor U. GroschelStewart, Zoologisches Institut, Darmstaclt, West Germany, for her generous gift of antibodies to tropomyosin and myosin. This work was supported by the National Heart Foundation of Australia.
References 1. 2.
3. 4.
5.
6.
AKESTER, A. R. Intercalated discs, nexuses, sarcoplasmic reticulum and transitional cells in the heart of the adult domestic fowl (Callus Gallus Domesticus). J Anat 133, 161-179 (1981). BENCOSME, S. A., TRILLO, A., ALANIS, J., BENITEZ, D. Correlative ultrastructural and electrophysiological study of the Purkinje system of the heart. J Electrocardiol2,27-28 ( 1969). CAESAR, R., EDWARDS, G. A., RUSKA, H. Electron microscopy of the impulse conducting system of the sheep heart. 2 Zellforsch Mikrosk Anat 48, 698-719 (1958). CHAMLEY, J. H., CAMPBELL, G. R., MCCONNELL, J. D., GRBSCHEL-STEWART, U. Comparison of vascular smooth muscle cells from adult human, monkey and rabbit in primary culture and in subculture. Cell Tissue Res 177,503-522 (1977). CHAMLEY-CAMPBELL, J. ‘H., CAMPBELI,, G. R., GRGSCHEL-STEWART, U., BURNSTOCK, G. FITC-labelled antibody staining of tropomyosin-containing fibrils in smooth, cardiac and skeletal muscle cells, pre-fusion myoblasts, fibroblasts, endothelial cells and 3T3 cells in culture. Cell Tissue Res 183, 153-166 (1977). CHAMLEY-CAMPBELL, J. H., CAMPBELL, G. R., Ross, R. The smooth muscle cell in culture. Physiol Rev 59, 1-61 (1979).
206 7.
E. Canale
G., FOZZARD, H. A. Effect of stretch on conduction velocity and cable properties of cardiac fibres. Am J Physio1237, Cl 19X124 (1979). ERIKSSON, A., THORNELL, L.-E. Intermediate (skeletin) filaments in heart Purkinje fibres. J Cell Biol 80, 231-247 (1979). GR~SCHEL-STEWART, U., CHAMLEY, J. H., MCCONNELL, J. D., BURNSTOCK, G. Comparison of the reaction of cultured smooth and cardiac muscle cells and fibroblasts to specific antibodies to myosin. Histochemistry 43, 215-224 (1975). GR~SCHEL-STEWART, U., SCHREIBER, J., MAHLMEISTER, C., WEBER, K. Production of specific antibodies to contractile proteins, and their use in immunofluorescence microscopy. I. Antibodies to smooth and striated chicken muscle myosins. Histochemie 46,229-236 (1976). LIPSIUS, S. L., FOZZARD, H. A., GIBBONS, W. R. Voltage and time dependence of restitution in heart. Am J Physio1243 (Heart Circ Physiol 12) H68-H76 (1982). Lrps~us, S. L., GIBBONS, W. R. Membrane currents, contractions and aftercontractions in cardiac Purkinje fibres. Am J Physiol 243 (Heart Circ Physiol 12) H77-H86 (1982). MARK, G. E., CHAMLEY, J. H., BURNSTOCK, G. Interactions between autonomic nerves and smooth and cardiac muscle cells in tissue culture. Dev Biol 32, 194-200 (1973). MASSON-P&ET, M., JONGSMA, H. J., BLEEKER, W. K., TSJERNINA, L., VAN GINNEKEN, A. C. G., TREI~L, B. W., BOUMAN, L. N. Intact isolated sinus node cells from the adult rabbit heart. J Moi Cell Cardiol 14, 295-299 ( 1982). MUIR, A. R. Observations on the fine structure of the Purkinje fibres in the ventricles of the sheeps heart. J Anat 91, 251-258 (1957). N~AEz-Du&, H. Electron microscopic study of the sarcolemma of Purkinje cells of the goat heart. Acta Anat 109,19-24 (1981). OLIPHANT, L. W., LOEWEN, R. D. Filament systems in Purkinje cells of sheep heart: Possible alteration of myofibrillogenesis. J Mol Cell Cardiol 8, 679-688 (1976). SAITO, K., TAMURA, Y., SAITO, M., MATSUMURA, K., NIKI, T., MORI, H. Comparison of the subunit compositions and ATPase activities of myosin in the myocardium and conduction system. J Mol Cell Cardiol 13, 331-322 (1981). SANDERS, R., MYERBURG, R. J,, GELBAND, H., BASSETT, A. L. Dissimilar length-tension relations of canine ventricular muscle and false tendon: Electrophysiologic alterations accompanying deformation. J Mel .Cell Cardiol l&209-219 (1979). SCOTT, T. M. The ultrastructure of ordinary and Purkinje cells of the fowl heart. J Anat 110, 259-273 (1971). SOMMER, J. R., JOHNSON, E. A. Cardiac muscle:’ A comparative study of Purkinje fibrcs and ventricular fibres. J Cell Biol 36, 497-526 (1968). SOMMER, J. R., JOHNSON, E. A. Purkinje fibres of the heart examined with the peroxidase reaction. J Cell Biol 37, 570-574 (1968). SOMMER, J. R., JOHNSON, E. A. Ultrastructure of cardiac muscle. In: Handbook of Physiology, vol. 1, section 2, “The Heart”, Eds. Berne, R. M., Sperelakis, N. American Physiological Society, pp. 113-186 (1979). THORNELL, L.-E. Myofilament-polyribosome complexes in the conducting system of hearts from cow, rabbit and cat. J Ultrastruct Res 41, 579-596 (1972). THORNELL, L.-E. Evidence of an imbalance in synthesis and degradation of myofibrillar proteins in rabbit Purkinje fibrcs. J Ultrastruct Rcs 44, 85-95 (1973). THORNELL, L.-E. Ultrastructural variations of Z-bands in cow Purkinje fibres. J Mol Cell Cardiol5,409-417 (1973). THORNELL, L.-E., ERIKSSON, A. Filament systems in the Purkinje fibres of the heart. Am J Physiol 241, H291-H305 (1981). THORNELL, L.-E., ERIKSSON, A., STIGBRAND, T., SJOSTROM, M. Structural proteins in cow Purkinje and ordinary ventricular fibres-a marked difference. J Mol Cell Cardiol 10,605-616 (1978). THORNELL, L.-E., SJOSTROM, M., ANDERSON, K.-E. The relationship between mechanical stress and myofibrillar organization in heart Purkinje fibres. J Mol Cell Cardio18, 689-695 (1976). VI~ACH, Sz., CHALLICE, C. E. Variations in filamentous and fibrillar organization, and associated sarcolemmal structures in cells of the normal mammalian heart. J Ultrastruct Res 28,321-334 (1969). DOMINGUEZ,
Purkinje
8. 9.
10.
11. 12. 13. 14.
15. 16. 17. 18.
19.
20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
et al.