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Effect of short dimethylsulfoxide perfusions on ultrastructure of the isolated rat heart
3inmal of Molecular
and Cellular
Cardiology
(1973)
5, 63-69
Effect of Short Dimethylsulfoxide on Ultrastructure of the Isolated D. FEUVRAY Luboratoire
(Received
de Physiologie
8
June 1972,
AND
Perfusions Rat Heart
J. DE LEIRIS
cornpark, Uniuersitt! Paris 91--ORSAY, France and accepted in revisedform
XI&-Centre
27 September
d’Orsay,
1972)
D. FEUVRAY AND J. DE LEIRIS. Effect of Short Dimethylsulfoxide Perfusions on Ultrastructure of the Isolated Rat Heart. Journal of Molecular and Cellular Cardiology (1973) 5, 63-69. Isolated rat hearts were perfused with dimethylsulfoxide (0.7 and 2.1 M) for 10 min at 37°C. The ventricular ultrastructure was studied (i) after 10 min of dimethylsulfoxide perfusion; and (ii) 2 min after replacement with normal solution following dimethylsulfoxide perfusion. Ultrastructural alterations were found after the dimethylsulfoxide perfusion was over, when returning the heart to the normal solution; changes were dependent on the dimethylsulfoxide concentration. The T-system and mitochondria were chiefly involved. A hyperosmotic effect of dimethylsulfoxide could be responsible
1. Introduction The biological actions of dimethylsulfoxide have attracted great interest for the past 10 years in view of the possible medical application of this compound. This interest was stimulated by the various applications of this drug in radioprotection, cryoprotection, membrane penetration and transport, anti-inflammation, nerve blockade (analgesia), and enhancement of the effectiveness of other drugs [13]. One of the important properties of dimethylsulfoxide is its cryoprotective action [I] and many studies have shown the effect of dimethylsulfoxide on living cells [23]. On cardiac tissue, moderate concentrations of dimethylsulfoxide (0.7 M to 2.1 M) reversibly modify mechanical activity, inducing large negative inotropic effect on whole isolated perfused heart [S, 191 but positive inotropic effect on isolated atria [3, 181. At these concentrations, dimethylsulfoxide also produces marked but reversible modifications of electrical activity [6]. These effects which indicate large alterations of cellular properties, may largely be attributed to a hyperosmotic effect [S]. At higher concentration (3 M), dimethylsulfoxide always induces an irreversible toxic effect [IO]. Another effect of dimethylsulfoxide on isolated
heart
is the
intracellular
release
of lactate
dehydrogenase
after
a perfusion
with dimethylsulfoxide when returning the heart to a normal perfusion medium [S, 151. The possible factors accounting for the latter effect are unknown but it is possible that it may be attributed to a direct action of dimethylsulfoxide on cell membrane since hyperosmotic perfusions did not produce enzyme release from
64
D.
FEUVRAY
AND
J. DE
LEIRIS
isolated heart [14]. Shlafer and Karow described ultrastructural alterations in rat ventricular myocardium after prolonged perfusions with dimethylsulfoxide [19] and suggestedthat these alterations could not be attributed to osmotic imbalances. The purpose of this report is to present results of ultrastructural studies of rat ventricular myocardium submitted to short periods of perfusion with dimethylsulfoxide and to determine whether ultrastructural alterations occurred during the dimethylsulfoxide perfusion or after returning to normal perfusing medium.
2. Materials
and
Methods
Young adult male Wistar rats weighing 150 to 200 g and fed according to the standard diet were used. The heart was excised from animals lightly anaesthetized with ether, and perfused at a constant flow (2.5 ml/min) through the coronary vessels.The heart was spontaneously beating. The temperature of the perfusion fluid was 37°C. The perfusion fluid had the following composition in mu: NaCl, 143; KCI, 5.6; CaCla, 2.16; MgClz, 0.24; Tris-(hydroxymethyl)aminomethane, 4.9; dextrose, 11. The pH was adjusted to 7.4 by addition of HCl 0.1 N. This solution was saturated with pure oxygen. Dimethylsulfoxide (Noury-Baker, N.V.; Deventer-Holland) was used at two concentrations : 0.7 M (5 %, v/v) and 2.1 M ( 15%, v/v). The osmolarity of the solutions were respectively: 330 mosmol (simple perfusate), 1050 mosmol (0.7 M-DMSO containing solution), 2450 mosmol (2.1 M-DMSO containing solution), 415 mosmol (fixative). In all our experiments hearts were initially perfused with the normal medium for a 20 min stabilization period. The perfusion medium containing dimethylsulfoxide was then substituted for the normal medium for a period of 10min. After this period, the heart was returned to the normal medium. Hearts were fixed for electron microscopic studies at various stages of this procedure: (i) after the stabilization period (control) ; (ii) at the end of the dimethylsulfoxide perfusion; (iii) 2 min after returning the heart to a normal medium after the dimethylsulfoxide removal, i.e. when enzyme releasewas at a maximum, as previously described. Immediately before the fixation, 0.1 y. procaine was added to the perfusion medium to produce complete cardiac relaxation. The fixative medium had the following composition : glutaraldehyde 2.5% in 0.1 M-Millonig’s phosphate buffer at pH 7.4 and at 4°C. After a 5 min perfusion with this medium, the heart was immersed in cold fixative; a portion of the right ventricle was minced into small pieces (about 1 mms) and allowed to fix in cold glutaraldehyde during 150 min. The tissue pieces were then rinsed in phosphate buffer for 12 h, washed several times in the same buffer and post-fixed in buffered osmic acid at 1% for 1 h. The specimenswere dehydrated in ethanol and embedded in Epon. Ultra-thin
L’L.ATl; I. Elcrtron micrograph of right ventricular muscle after 20 min of normal perfusiorl ic ontrol). This longitudinal section illustrates dense mitochondria (M), transverse tubulrs /‘I‘.. \arcoplasmic rrticulurn (SRI, adjacmt cistern of sarroplasmic wticulum (Cl) and basement mcr!~'4 000. 1,I a,,, I hm I. 1.f;?ciq ,tMIc'P (1-l )
PLATE 2. Section of right ventricular sulfoxide. No detectable changes appear droplet. Y 27500.
muscle fixed after 10 min of perfusion in mitochondria (M) and transwrse
with 0.7 wdimethyltubules (T). L: lipid
PLATE 3. Electron micrograph ‘;ullixicle perfusion. Note the slight
of right swelling
ventricular of some
muscle T-tubulrs
fixed (T).
after 10 min . 36000.
of 2.1 M-dimethyl-
Cm. lumen
PLATE 4. Section of right ventricular following 10 min of 0.7 wdimethylsulfoxide. (I) are observed. Mitorhondria appear
muscle fixed 2 min after replacement Swelling of some T-tubules (T) normal. ‘j’ 15000.
with normal and interstitial
solution oedema
PLATE following srparation dcumosomr:
5. Section of right ventricular muscle fixed 2 min after replacement 10 min of 2.1 wdimethylsulfoxide. Note the marked mitochondrinl of cristae and some membrane disruptions. Swollen ‘L‘-tubulrr (‘I‘\ rndo. : endothrlium ; bm: basrmrnt mrmhranv. IHIK~O.
with normal solutiwr (M) sw-riling x\ it h arr
also
vi\il+.
(It.\
PLATE 6. This plate illustrates tubules (Tb) and normal T-tubules
the variability of ultrastructural (Ta) are observed. x 11000.
modifications.
Dilated
T-
DIMETHYLSULFOXIDE
AND
ULTRASTRUCTURE
OF ISOLATED
sections were cut on a LKB ultramicrotome, stained acetate and examined with a Siemens Elmiskop 1A.
with
65
HEART
lead citrate
and uranyl
3. Results Control experiments The ultrastructure of the ventricular myocardial cells from the isolated rat hearts perfused with normal conditions during 20 min stabilization period is illustrated in Plate 1. This plate shows the typical general morphology with good preservation of ultrastructure. Myofibrillar structure and A-bands or I-bands are normal. Mitochondria appear dense with numerous well preserved cristae. Sarcoplasmic reticulum and T-tubules are normal in size and configuration. Other micrographs also show that interstitial spaces are not swollen. Hearts after 10 min perfusion with 0.7
M
-dimethylsulfoxide
After 10 min of 0.7 M-dimethylsulfoxide perfusion, the morphology was generally normal in appearance as illustrated by the Plate 2. However, a moderate swelling of the interstitial spaces and of the T-tubules appeared in some hearts. MyofibriIlar structure, mitochondria and sarcoplasmic reticulum remained normal.
Hearts after 10 min perfusion with 2.1 M-dimethylsulfoxide As in the previous case, the morphology dimethylsulfoxide perfusion. However appeared as shown in Plate 3; in other frequently observed.
is only slightly modified more frequent swelling micrographs, interstitial
at the end of the of the T-system oedema was also
Hearts 2 min after replacement with normal solution following 10 min of 0.7 M-dimethylsulfoxide The myofibrils and mitochondria appeared normal. T-tubules were frequently swollen and their membrane was sometimes disrupted while the sarcoplasmic reticulum was not altered. Interstitial oedema was generally present (Plate 4). Hearts 2 min after replacement with normal solution following 10 min of 2. I M-dirnethylsuljoxide Striking changes were observed (Plate 5). Many mitochondria appeared swollen with separation of cristae, and in some cases their membranes were disrupted. The myofibrils and their cross-banding were not altered but sometimes they were
66
D.
FEUVRAY
AND
J. DE
LEIRIS
contorted by the swelling of the mitochondria. There was always an extracellular oedema with marked swelling of transverse tubules and of interstitial spaces. These modifications were larger in some parts of the section than in others as seen in Plate 6. Altered mitochondria were observed most frequently near the swollen T-tubes. In areas with few mitochondria T-tubes appeared generally normal. In some cases the nuclear membrane was contorted and chromatin had a condensed configuration. The effects of dimethylsulfoxide on the ultrastructure varied from heart to heart.
4. Discussion Control hearts perfused during the 20 min stabilization period with normal medium presented no alterations of ultrastructure. Thus the isolated heart preparation maintains its ultrastructural integrity in our experimental conditions. Moreover, longer perfusion periods do not alter the structural integrity of this preparation. In our experiments the beating heart is arrested with procaine [I71 before fixation to prevent the heart tissue from contracting on contact with the fixative, which would prevent the uniform distribution of fixative throughout the tissue [S, 161. After an abrupt removal of dimethylsulfoxide upon returning the heart to a normal perfusion, striking ultrastructural modifications appear in agreement with Shlafer and Karow [19]. But during the dimethylsulfoxide perfusion, we never observed large changes. So it seems probable that the observed ultrastructural alterations are not directly related to the dimethylsulfoxide effect but more likely to the removal of this substance. Niemeyer and Forssmann [16] observed drastic morphological changes after removal of hypertonic solutions of glycerol when frog skeletal muscle (but not mammalian heart) was returned to normal Ringer solution. The osmolarity of the dimethylsulfoxide solutions particularly at 2.1 M appears quite high but this substance diffuses rapidly across the membrane. It is likely that the osmotic stress resulting from the beginning of the perfusion by the dimethylsulfoxide-containing medium diminished rapidly with time as the cellular uptake of dimethylsulfoxide increased. It is likely that when returning the heart to the normal perfusion the removal of dimethylsulfoxide resulted in a large transmembrane concentration gradient which produced a major uptake of water. Using the barnacle muscle, Bunch and Edwards [Z] indicated that the cell was unable to concentrate dimethylsulfoxide and that after 10 min of bathing dimethylsulfoxide solution, the dimethylsulfoxide concentration into the cell was about 30% of the extracellular concentration. If this value is taken to be the same for rat heart, we can assume that at the end of the dimethylsulfoxide perfusion, the concentrations of this substance may be 100% in the extracellular compartment, particularly in the T-tubes, and about 30% inside the cell. The marked swelling of the T-tubules could be explained as follows. If T-tubules constitute relatively closed and tortuous
DIMETHYLSULFOXIDE
AND
ULTRASTRUCTURE
OF ISOLATED
HEART
67
areas [7j, a larger osmotic gradient would develop between the T-tubes and the external medium than between the cell and the extracellular compartment upon returning the heart to the normal medium. This dilatation probably diminished a few minutes after removal of extracellular dimethylsulfoxide which would agree with the observations of Shlafer and Karow [19] who did not find as large a swelling of T-tubules as those shown in our micrographs 20 min after removal of dimethyls&oxide. Such a hypothesis is not in agreement with the observations of Strosberg et al. [21] on guinea-pig myocardium. They found very slight modifications of T-system after replacement with normal solution following treatment with hypertonic glycerol solution. However, the T-tubules of guinea-pig are very wide [4] and it is likely that osmotic gradient between T-tubes and extracellular medium would be much weaker in this species than in rat ventricular myocardium. Our observations are in full agreement with the assumption of Howell and Jenden [12J who showed that skeletal muscle cells placed in hypertonic solutions shrank and on return to Ringer swelled as a result of large water movements. These investigators observed disruptions of the T-tubes when muscles were exposed to normal Ringer following treatment with 400 mM-glycerol and concluded that osmotic phenomenon was involved in these disruptions of T-system. The condensed configuration of chromatin observed in a few micrographs can also be attributed to an osmotic effect of dimethylsulfoxide [16]. Dreifuss et al. [.5j suggested that dilatation of the T-tubules under the effect of hypertonic perfusions could explain the decrease in conduction speed. The increase in the latency of the action potential appearing under the effect of dimethylsulfoxide [S] could be related to such a modification of the T-tubes. We found marked changes in mitochondria after changing back from 2.1 Mdimethylsulfoxide to a normal perfusion. Similar modifications of mitochondria have been already described by Strosberg et al. on papillary guinea-pig muscle after removal of glycerol [2Z]. Shlafer and Karow [19] also described swelling of mitochondria after 2.1 to 2.82 M-dimethylsulfoxide. The fact that alterations in mitochondria appear near the swollen T-tubes is not surprising because Girardier et al. [9] reported a special relationship between these two structures. After dimethylsulfoxide perfusions, there is release of lactate dehydrogenase from isolated rat heart [S, 151. Such a release is not induced by hypertonic perfusions with sucrose [14] although these perfusions produced large swelling of the axial-transverse system [20]. This last observation is not in disagreement with the hypothesis that the dimethylsulfoxide effect is due to the hyperosmolarity of the solution. Indeed, the cellular effects of sucrose are evidently different since dimethylsulfoxide can easily penetrate into the cell while sucrose does not. It is likely that the lactate dehydrogenase release appearing after the dimethylsulfoxide removal is directly related to the observed alterations of ultrastructure. The reasons for the variability of ultrastructural modifications encountered under the effect of dimethvlsulfoxide from heart to heart and even from fibre to fibre
68
D.
FEUVRAY
AND
J. DE
LEIRIS
are not clear. It is difficult to imagine that the penetration of the abnormal medium or the water movement can be very different in areas of the same cell which are so close to each other. Many authors have postulated a direct effect of dimethylsulfoxide [II, 191 on the cell membrane. Our observations do not support entirely this assumption. Indeed, such an effect would increase with time during the dimethylsulfoxide perfusion and ultrastructural alterations would be maximum at the end of the dimethylsulfoxide perfusion. It is more likely that the hyperosmolarity of the dimethylsulfoxide solutions and the osmotic stress induced by the removal of this substance are responsible for the ultrastructural alterations observed. Acknowledgments Our thanks are due to Professor E. Coraboeuf for helpful suggestions. We deeply appreciate the critical advice of Professor L. Girardier during the conduct of this work. REFERENCES 1. 2.
M. J. Radioprotective and cryoprotective properties of dimethylin cellular systems. Annals of the .New York Academy of Sciences 141, 45-62
i%HwOOD-SbfITH,
sulfoxide (1967).
W. & EDWARDS, C. The permeation of non-electrolytes through the single muscle cell. j%urnal of Physiology, London 202, 683-697 (1969). R. A., BLACKBURN, K. J. & SPILKER, B. A. Effects of dimethylsulfoxide, 3. BURGES, dimethylformamide and dimethylacetamide on myocardial contractility and enzyme activity. Life Sciences 8, 1325-1335 (1969). 4. DENOIT, F. & CORABOEUP, E. Etude comparative de l’ultrastructure du myocarde chez le Rat et le Cobaye. Comptes Rendus des Slances de la So&! de Biologic et de sesFiliah 159,211&2121 (1965). 5. DREIFUSS, J. J., GIRARDIER, L. & FORSSMANN,W. G. Etude delapropagationde l’excitation dans le ventricule de Rat au moyen de solutions hypertoniques. @iigers Archiv ftir die gesamte Plpiologie des Men&x und der Tiere 292, 13-33 (1966). 6. FE-Y, D. & DE LEms, J. Effect of dimethylsulloxide on isolated rat heart and lacticodehydrogenase release. Eurohean Journal of Pharmacolo~ 16, 8-13 (1971). 7. FORSSMANN, W. G. & GIRAIUXER, L. A study of the T-system in rat heart. Ihe Journal of Cell Biology 44, 1-19 (1970). 8. FORSSMANN, W. G., SIEGRIST, G., ORCI, L., GIRARDLER, L., PICKET, R. & ROUILLER, C. Fixation par perfusion pour la microscopic electronique. Essai de gtntralisation. Journal & Microscopic 6,279-304 (1967). 9. GIRARDIER, L., Droswuss, J. J., HAENNI, B. & PETROVICI, A. Reponse du tissu myocardique de rat in vitro a une augmentation de la pression osmotique du milieu externe. Pathologia et Mitrobiologia 27, 16-30 (1964). 10. HOBBS, K. E. F. & HUGGINS, C. E. Investigation of the effects of cryophylactic agents on the isolated rat heart at 37°C. Cryobiolosy 6, 239-245 (1969). 11. HOLLAND, W. C. & PORTER, M. T. Pharmacological effects of drugs on excitationcontraction coupling in cardiac muscle. Federation Proceedings. Federation of American Societiesfor Experimental Biology 28, 1663-1669 (1969). BUNCH,
barnacle
DIMETHYLSULFOXIDE 12.
13. 14.
15. 16. 17.
18.
19.
20.
21.
AND
ULTRASTRUCTURE
OF ISOLATED
HEART
69
HOM~ELL, J. N. & JENDEN, D. J. T-tubules of skeletal muscle: morphological alterations which interrupt excitation-contraction coupling. Federation Proceedings. Federation of American Societies for Experimental Biology 26, 553 (1967). JACOB, S. W., ROSENBALJM, E. E. & WOOD, D. C. Dimethylsulfoxide l-Basic concepts of dimethylsulfoxide. New York: M. Dekker, Inc. (1971). DE LEIRIS, J., BRETON, D., FEUVRAY, D. & CORABOEUF, E. Lacticodehydrogenase release from perfused rat heart under the effect of abnormal media. Archives Internationales de Physiologie et de Biochimie 77, 749-762 (1969). DE LEIRIS, J., FEUVRAY, D., DIACONO, J. & DIETRICH, J. Action du dimtthylsulfoxyde sur le coeur isole et perfuse de Rat. Journal de Physiologic, Paris 61, 338-339 (1969). NIEMEYER, G. & FORSSMANN, W. G. Comparison of glycerol treatment in frog skeletal muscle and mammalian heart. Tlze 3ournal of Cell Biology 50, 288-299 (1971). ROY, P. E., GAILIS, L., MORIN, P. J. & COTE, G. A new beating heart preparation: ultrastructural studies with special reference to vesiculation of intercalated discs. Cardiovascular Research 5, 178-193 (1971). Sms, W. M., TROLL, N. V. & CRANTZ, P. L. The effect of dimethylsulfoxide on isolated-innervated skeletal, smooth and cardiac muscle. Proceedings of the Society for Expere’mental Biology and Medicine 122, 103-107 (1966). SHLAFER, M. & KARow, A. M. Ultrastructure-function correlative studies for cardiac cryopreservation. I-Hearts perfused with various concentrations of dimethylsulfoxide. Cpobiology 8,280-289 (1971). SPERELAKIS, N. & RUBIO, R. An orderly lattice of axial tubules which interconnect adjacent transverse tubules in guinea-pig ventricular myocardium. Journal of Molecular and Cellular Cardiology 2,212-220 (1971). STROSBERG, A. M., KATZUNG, B. G. & LEE, J. C. Glycerol removal treatment of guinea-pig cardiac muscle. Journal of Molecular and Cellular Cardiology 4, 39-48 (1972).
and Cellular
Cardiology
(1973)
5, 63-69
Effect of Short Dimethylsulfoxide on Ultrastructure of the Isolated D. FEUVRAY Luboratoire
(Received
de Physiologie
8
June 1972,
AND
Perfusions Rat Heart
J. DE LEIRIS
cornpark, Uniuersitt! Paris 91--ORSAY, France and accepted in revisedform
XI&-Centre
27 September
d’Orsay,
1972)
D. FEUVRAY AND J. DE LEIRIS. Effect of Short Dimethylsulfoxide Perfusions on Ultrastructure of the Isolated Rat Heart. Journal of Molecular and Cellular Cardiology (1973) 5, 63-69. Isolated rat hearts were perfused with dimethylsulfoxide (0.7 and 2.1 M) for 10 min at 37°C. The ventricular ultrastructure was studied (i) after 10 min of dimethylsulfoxide perfusion; and (ii) 2 min after replacement with normal solution following dimethylsulfoxide perfusion. Ultrastructural alterations were found after the dimethylsulfoxide perfusion was over, when returning the heart to the normal solution; changes were dependent on the dimethylsulfoxide concentration. The T-system and mitochondria were chiefly involved. A hyperosmotic effect of dimethylsulfoxide could be responsible
1. Introduction The biological actions of dimethylsulfoxide have attracted great interest for the past 10 years in view of the possible medical application of this compound. This interest was stimulated by the various applications of this drug in radioprotection, cryoprotection, membrane penetration and transport, anti-inflammation, nerve blockade (analgesia), and enhancement of the effectiveness of other drugs [13]. One of the important properties of dimethylsulfoxide is its cryoprotective action [I] and many studies have shown the effect of dimethylsulfoxide on living cells [23]. On cardiac tissue, moderate concentrations of dimethylsulfoxide (0.7 M to 2.1 M) reversibly modify mechanical activity, inducing large negative inotropic effect on whole isolated perfused heart [S, 191 but positive inotropic effect on isolated atria [3, 181. At these concentrations, dimethylsulfoxide also produces marked but reversible modifications of electrical activity [6]. These effects which indicate large alterations of cellular properties, may largely be attributed to a hyperosmotic effect [S]. At higher concentration (3 M), dimethylsulfoxide always induces an irreversible toxic effect [IO]. Another effect of dimethylsulfoxide on isolated
heart
is the
intracellular
release
of lactate
dehydrogenase
after
a perfusion
with dimethylsulfoxide when returning the heart to a normal perfusion medium [S, 151. The possible factors accounting for the latter effect are unknown but it is possible that it may be attributed to a direct action of dimethylsulfoxide on cell membrane since hyperosmotic perfusions did not produce enzyme release from
64
D.
FEUVRAY
AND
J. DE
LEIRIS
isolated heart [14]. Shlafer and Karow described ultrastructural alterations in rat ventricular myocardium after prolonged perfusions with dimethylsulfoxide [19] and suggestedthat these alterations could not be attributed to osmotic imbalances. The purpose of this report is to present results of ultrastructural studies of rat ventricular myocardium submitted to short periods of perfusion with dimethylsulfoxide and to determine whether ultrastructural alterations occurred during the dimethylsulfoxide perfusion or after returning to normal perfusing medium.
2. Materials
and
Methods
Young adult male Wistar rats weighing 150 to 200 g and fed according to the standard diet were used. The heart was excised from animals lightly anaesthetized with ether, and perfused at a constant flow (2.5 ml/min) through the coronary vessels.The heart was spontaneously beating. The temperature of the perfusion fluid was 37°C. The perfusion fluid had the following composition in mu: NaCl, 143; KCI, 5.6; CaCla, 2.16; MgClz, 0.24; Tris-(hydroxymethyl)aminomethane, 4.9; dextrose, 11. The pH was adjusted to 7.4 by addition of HCl 0.1 N. This solution was saturated with pure oxygen. Dimethylsulfoxide (Noury-Baker, N.V.; Deventer-Holland) was used at two concentrations : 0.7 M (5 %, v/v) and 2.1 M ( 15%, v/v). The osmolarity of the solutions were respectively: 330 mosmol (simple perfusate), 1050 mosmol (0.7 M-DMSO containing solution), 2450 mosmol (2.1 M-DMSO containing solution), 415 mosmol (fixative). In all our experiments hearts were initially perfused with the normal medium for a 20 min stabilization period. The perfusion medium containing dimethylsulfoxide was then substituted for the normal medium for a period of 10min. After this period, the heart was returned to the normal medium. Hearts were fixed for electron microscopic studies at various stages of this procedure: (i) after the stabilization period (control) ; (ii) at the end of the dimethylsulfoxide perfusion; (iii) 2 min after returning the heart to a normal medium after the dimethylsulfoxide removal, i.e. when enzyme releasewas at a maximum, as previously described. Immediately before the fixation, 0.1 y. procaine was added to the perfusion medium to produce complete cardiac relaxation. The fixative medium had the following composition : glutaraldehyde 2.5% in 0.1 M-Millonig’s phosphate buffer at pH 7.4 and at 4°C. After a 5 min perfusion with this medium, the heart was immersed in cold fixative; a portion of the right ventricle was minced into small pieces (about 1 mms) and allowed to fix in cold glutaraldehyde during 150 min. The tissue pieces were then rinsed in phosphate buffer for 12 h, washed several times in the same buffer and post-fixed in buffered osmic acid at 1% for 1 h. The specimenswere dehydrated in ethanol and embedded in Epon. Ultra-thin
L’L.ATl; I. Elcrtron micrograph of right ventricular muscle after 20 min of normal perfusiorl ic ontrol). This longitudinal section illustrates dense mitochondria (M), transverse tubulrs /‘I‘.. \arcoplasmic rrticulurn (SRI, adjacmt cistern of sarroplasmic wticulum (Cl) and basement mcr!~'4 000. 1,I a,,, I hm I. 1.f;?ciq ,tMIc'P (1-l )
PLATE 2. Section of right ventricular sulfoxide. No detectable changes appear droplet. Y 27500.
muscle fixed after 10 min of perfusion in mitochondria (M) and transwrse
with 0.7 wdimethyltubules (T). L: lipid
PLATE 3. Electron micrograph ‘;ullixicle perfusion. Note the slight
of right swelling
ventricular of some
muscle T-tubulrs
fixed (T).
after 10 min . 36000.
of 2.1 M-dimethyl-
Cm. lumen
PLATE 4. Section of right ventricular following 10 min of 0.7 wdimethylsulfoxide. (I) are observed. Mitorhondria appear
muscle fixed 2 min after replacement Swelling of some T-tubules (T) normal. ‘j’ 15000.
with normal and interstitial
solution oedema
PLATE following srparation dcumosomr:
5. Section of right ventricular muscle fixed 2 min after replacement 10 min of 2.1 wdimethylsulfoxide. Note the marked mitochondrinl of cristae and some membrane disruptions. Swollen ‘L‘-tubulrr (‘I‘\ rndo. : endothrlium ; bm: basrmrnt mrmhranv. IHIK~O.
with normal solutiwr (M) sw-riling x\ it h arr
also
vi\il+.
(It.\
PLATE 6. This plate illustrates tubules (Tb) and normal T-tubules
the variability of ultrastructural (Ta) are observed. x 11000.
modifications.
Dilated
T-
DIMETHYLSULFOXIDE
AND
ULTRASTRUCTURE
OF ISOLATED
sections were cut on a LKB ultramicrotome, stained acetate and examined with a Siemens Elmiskop 1A.
with
65
HEART
lead citrate
and uranyl
3. Results Control experiments The ultrastructure of the ventricular myocardial cells from the isolated rat hearts perfused with normal conditions during 20 min stabilization period is illustrated in Plate 1. This plate shows the typical general morphology with good preservation of ultrastructure. Myofibrillar structure and A-bands or I-bands are normal. Mitochondria appear dense with numerous well preserved cristae. Sarcoplasmic reticulum and T-tubules are normal in size and configuration. Other micrographs also show that interstitial spaces are not swollen. Hearts after 10 min perfusion with 0.7
M
-dimethylsulfoxide
After 10 min of 0.7 M-dimethylsulfoxide perfusion, the morphology was generally normal in appearance as illustrated by the Plate 2. However, a moderate swelling of the interstitial spaces and of the T-tubules appeared in some hearts. MyofibriIlar structure, mitochondria and sarcoplasmic reticulum remained normal.
Hearts after 10 min perfusion with 2.1 M-dimethylsulfoxide As in the previous case, the morphology dimethylsulfoxide perfusion. However appeared as shown in Plate 3; in other frequently observed.
is only slightly modified more frequent swelling micrographs, interstitial
at the end of the of the T-system oedema was also
Hearts 2 min after replacement with normal solution following 10 min of 0.7 M-dimethylsulfoxide The myofibrils and mitochondria appeared normal. T-tubules were frequently swollen and their membrane was sometimes disrupted while the sarcoplasmic reticulum was not altered. Interstitial oedema was generally present (Plate 4). Hearts 2 min after replacement with normal solution following 10 min of 2. I M-dirnethylsuljoxide Striking changes were observed (Plate 5). Many mitochondria appeared swollen with separation of cristae, and in some cases their membranes were disrupted. The myofibrils and their cross-banding were not altered but sometimes they were
66
D.
FEUVRAY
AND
J. DE
LEIRIS
contorted by the swelling of the mitochondria. There was always an extracellular oedema with marked swelling of transverse tubules and of interstitial spaces. These modifications were larger in some parts of the section than in others as seen in Plate 6. Altered mitochondria were observed most frequently near the swollen T-tubes. In areas with few mitochondria T-tubes appeared generally normal. In some cases the nuclear membrane was contorted and chromatin had a condensed configuration. The effects of dimethylsulfoxide on the ultrastructure varied from heart to heart.
4. Discussion Control hearts perfused during the 20 min stabilization period with normal medium presented no alterations of ultrastructure. Thus the isolated heart preparation maintains its ultrastructural integrity in our experimental conditions. Moreover, longer perfusion periods do not alter the structural integrity of this preparation. In our experiments the beating heart is arrested with procaine [I71 before fixation to prevent the heart tissue from contracting on contact with the fixative, which would prevent the uniform distribution of fixative throughout the tissue [S, 161. After an abrupt removal of dimethylsulfoxide upon returning the heart to a normal perfusion, striking ultrastructural modifications appear in agreement with Shlafer and Karow [19]. But during the dimethylsulfoxide perfusion, we never observed large changes. So it seems probable that the observed ultrastructural alterations are not directly related to the dimethylsulfoxide effect but more likely to the removal of this substance. Niemeyer and Forssmann [16] observed drastic morphological changes after removal of hypertonic solutions of glycerol when frog skeletal muscle (but not mammalian heart) was returned to normal Ringer solution. The osmolarity of the dimethylsulfoxide solutions particularly at 2.1 M appears quite high but this substance diffuses rapidly across the membrane. It is likely that the osmotic stress resulting from the beginning of the perfusion by the dimethylsulfoxide-containing medium diminished rapidly with time as the cellular uptake of dimethylsulfoxide increased. It is likely that when returning the heart to the normal perfusion the removal of dimethylsulfoxide resulted in a large transmembrane concentration gradient which produced a major uptake of water. Using the barnacle muscle, Bunch and Edwards [Z] indicated that the cell was unable to concentrate dimethylsulfoxide and that after 10 min of bathing dimethylsulfoxide solution, the dimethylsulfoxide concentration into the cell was about 30% of the extracellular concentration. If this value is taken to be the same for rat heart, we can assume that at the end of the dimethylsulfoxide perfusion, the concentrations of this substance may be 100% in the extracellular compartment, particularly in the T-tubes, and about 30% inside the cell. The marked swelling of the T-tubules could be explained as follows. If T-tubules constitute relatively closed and tortuous
DIMETHYLSULFOXIDE
AND
ULTRASTRUCTURE
OF ISOLATED
HEART
67
areas [7j, a larger osmotic gradient would develop between the T-tubes and the external medium than between the cell and the extracellular compartment upon returning the heart to the normal medium. This dilatation probably diminished a few minutes after removal of extracellular dimethylsulfoxide which would agree with the observations of Shlafer and Karow [19] who did not find as large a swelling of T-tubules as those shown in our micrographs 20 min after removal of dimethyls&oxide. Such a hypothesis is not in agreement with the observations of Strosberg et al. [21] on guinea-pig myocardium. They found very slight modifications of T-system after replacement with normal solution following treatment with hypertonic glycerol solution. However, the T-tubules of guinea-pig are very wide [4] and it is likely that osmotic gradient between T-tubes and extracellular medium would be much weaker in this species than in rat ventricular myocardium. Our observations are in full agreement with the assumption of Howell and Jenden [12J who showed that skeletal muscle cells placed in hypertonic solutions shrank and on return to Ringer swelled as a result of large water movements. These investigators observed disruptions of the T-tubes when muscles were exposed to normal Ringer following treatment with 400 mM-glycerol and concluded that osmotic phenomenon was involved in these disruptions of T-system. The condensed configuration of chromatin observed in a few micrographs can also be attributed to an osmotic effect of dimethylsulfoxide [16]. Dreifuss et al. [.5j suggested that dilatation of the T-tubules under the effect of hypertonic perfusions could explain the decrease in conduction speed. The increase in the latency of the action potential appearing under the effect of dimethylsulfoxide [S] could be related to such a modification of the T-tubes. We found marked changes in mitochondria after changing back from 2.1 Mdimethylsulfoxide to a normal perfusion. Similar modifications of mitochondria have been already described by Strosberg et al. on papillary guinea-pig muscle after removal of glycerol [2Z]. Shlafer and Karow [19] also described swelling of mitochondria after 2.1 to 2.82 M-dimethylsulfoxide. The fact that alterations in mitochondria appear near the swollen T-tubes is not surprising because Girardier et al. [9] reported a special relationship between these two structures. After dimethylsulfoxide perfusions, there is release of lactate dehydrogenase from isolated rat heart [S, 151. Such a release is not induced by hypertonic perfusions with sucrose [14] although these perfusions produced large swelling of the axial-transverse system [20]. This last observation is not in disagreement with the hypothesis that the dimethylsulfoxide effect is due to the hyperosmolarity of the solution. Indeed, the cellular effects of sucrose are evidently different since dimethylsulfoxide can easily penetrate into the cell while sucrose does not. It is likely that the lactate dehydrogenase release appearing after the dimethylsulfoxide removal is directly related to the observed alterations of ultrastructure. The reasons for the variability of ultrastructural modifications encountered under the effect of dimethvlsulfoxide from heart to heart and even from fibre to fibre
68
D.
FEUVRAY
AND
J. DE
LEIRIS
are not clear. It is difficult to imagine that the penetration of the abnormal medium or the water movement can be very different in areas of the same cell which are so close to each other. Many authors have postulated a direct effect of dimethylsulfoxide [II, 191 on the cell membrane. Our observations do not support entirely this assumption. Indeed, such an effect would increase with time during the dimethylsulfoxide perfusion and ultrastructural alterations would be maximum at the end of the dimethylsulfoxide perfusion. It is more likely that the hyperosmolarity of the dimethylsulfoxide solutions and the osmotic stress induced by the removal of this substance are responsible for the ultrastructural alterations observed. Acknowledgments Our thanks are due to Professor E. Coraboeuf for helpful suggestions. We deeply appreciate the critical advice of Professor L. Girardier during the conduct of this work. REFERENCES 1. 2.
M. J. Radioprotective and cryoprotective properties of dimethylin cellular systems. Annals of the .New York Academy of Sciences 141, 45-62
i%HwOOD-SbfITH,
sulfoxide (1967).
W. & EDWARDS, C. The permeation of non-electrolytes through the single muscle cell. j%urnal of Physiology, London 202, 683-697 (1969). R. A., BLACKBURN, K. J. & SPILKER, B. A. Effects of dimethylsulfoxide, 3. BURGES, dimethylformamide and dimethylacetamide on myocardial contractility and enzyme activity. Life Sciences 8, 1325-1335 (1969). 4. DENOIT, F. & CORABOEUP, E. Etude comparative de l’ultrastructure du myocarde chez le Rat et le Cobaye. Comptes Rendus des Slances de la So&! de Biologic et de sesFiliah 159,211&2121 (1965). 5. DREIFUSS, J. J., GIRARDIER, L. & FORSSMANN,W. G. Etude delapropagationde l’excitation dans le ventricule de Rat au moyen de solutions hypertoniques. @iigers Archiv ftir die gesamte Plpiologie des Men&x und der Tiere 292, 13-33 (1966). 6. FE-Y, D. & DE LEms, J. Effect of dimethylsulloxide on isolated rat heart and lacticodehydrogenase release. Eurohean Journal of Pharmacolo~ 16, 8-13 (1971). 7. FORSSMANN, W. G. & GIRAIUXER, L. A study of the T-system in rat heart. Ihe Journal of Cell Biology 44, 1-19 (1970). 8. FORSSMANN, W. G., SIEGRIST, G., ORCI, L., GIRARDLER, L., PICKET, R. & ROUILLER, C. Fixation par perfusion pour la microscopic electronique. Essai de gtntralisation. Journal & Microscopic 6,279-304 (1967). 9. GIRARDIER, L., Droswuss, J. J., HAENNI, B. & PETROVICI, A. Reponse du tissu myocardique de rat in vitro a une augmentation de la pression osmotique du milieu externe. Pathologia et Mitrobiologia 27, 16-30 (1964). 10. HOBBS, K. E. F. & HUGGINS, C. E. Investigation of the effects of cryophylactic agents on the isolated rat heart at 37°C. Cryobiolosy 6, 239-245 (1969). 11. HOLLAND, W. C. & PORTER, M. T. Pharmacological effects of drugs on excitationcontraction coupling in cardiac muscle. Federation Proceedings. Federation of American Societiesfor Experimental Biology 28, 1663-1669 (1969). BUNCH,
barnacle
DIMETHYLSULFOXIDE 12.
13. 14.
15. 16. 17.
18.
19.
20.
21.
AND
ULTRASTRUCTURE
OF ISOLATED
HEART
69
HOM~ELL, J. N. & JENDEN, D. J. T-tubules of skeletal muscle: morphological alterations which interrupt excitation-contraction coupling. Federation Proceedings. Federation of American Societies for Experimental Biology 26, 553 (1967). JACOB, S. W., ROSENBALJM, E. E. & WOOD, D. C. Dimethylsulfoxide l-Basic concepts of dimethylsulfoxide. New York: M. Dekker, Inc. (1971). DE LEIRIS, J., BRETON, D., FEUVRAY, D. & CORABOEUF, E. Lacticodehydrogenase release from perfused rat heart under the effect of abnormal media. Archives Internationales de Physiologie et de Biochimie 77, 749-762 (1969). DE LEIRIS, J., FEUVRAY, D., DIACONO, J. & DIETRICH, J. Action du dimtthylsulfoxyde sur le coeur isole et perfuse de Rat. Journal de Physiologic, Paris 61, 338-339 (1969). NIEMEYER, G. & FORSSMANN, W. G. Comparison of glycerol treatment in frog skeletal muscle and mammalian heart. Tlze 3ournal of Cell Biology 50, 288-299 (1971). ROY, P. E., GAILIS, L., MORIN, P. J. & COTE, G. A new beating heart preparation: ultrastructural studies with special reference to vesiculation of intercalated discs. Cardiovascular Research 5, 178-193 (1971). Sms, W. M., TROLL, N. V. & CRANTZ, P. L. The effect of dimethylsulfoxide on isolated-innervated skeletal, smooth and cardiac muscle. Proceedings of the Society for Expere’mental Biology and Medicine 122, 103-107 (1966). SHLAFER, M. & KARow, A. M. Ultrastructure-function correlative studies for cardiac cryopreservation. I-Hearts perfused with various concentrations of dimethylsulfoxide. Cpobiology 8,280-289 (1971). SPERELAKIS, N. & RUBIO, R. An orderly lattice of axial tubules which interconnect adjacent transverse tubules in guinea-pig ventricular myocardium. Journal of Molecular and Cellular Cardiology 2,212-220 (1971). STROSBERG, A. M., KATZUNG, B. G. & LEE, J. C. Glycerol removal treatment of guinea-pig cardiac muscle. Journal of Molecular and Cellular Cardiology 4, 39-48 (1972).