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Sarcolemmal disruption during the calcium paradox
J Mol Cell Cardiol 17, 265 273 (1985)
Sarcolemmal
Disruption During the Calcium Paradox
j. A. Post, P. F. E. M. Nievelstein, J. Leunissen-Bijvelt, A. J. Verkleij* and T. J. C. Rulgrok~ Department of Molecular Cell Biology, and *Institute of Molecular Biology, State University of Utrecht, Utrecht, The Netherlands and t Department of Cardiology, University Hospital, Utrecht, The Netherlands (Received 1 February 1984, accepted in revisedform 21 August 1984) j. A. POST,P. F. E. M. NIEVELSTEIN,J. LEUNISSEN-BIJVELT,A.J.
VERKLEIJ AND T.J.C. RUIGROK.Sarcolemmal Disruption During the Calcimn Paradox. Journalof Molecularand CellularCardiology(1985) 17, 265 273. Reperfusion of an isolated heart with calcium-containing solution after a short period of calcium-free perfusion may result in irreversible cell damage (calcium paradox). The ultrastructure of the sarcolemma of the rabbit heart during the calcium paradox was studied by using fast freezing devices. This method excluded ultrastructural changes induced by chemical fixation and cryoprotection. In addition, thin-section and conventional freezefracture electron microscopy were used. During reperfusion with calcium-containing solution disruption of the sarcolemma was observed, which was attended with formation of unilamellar and multilamellar vesicles and aggregation of intramembrane particles. These ultrastructural changes are explained in terms of calcium- and proton-induced lateral phase separation and fusion processesin the lipid bilayer of the sarcolemma.
KEY WORDS: Calcium paradox; Rabbit hearts; Freeze-fracture electron microscopy; Fast freezing devices; Sarcolemma ; Phospholipids ; Vesiculation; Intramembrane particles.
Introduction R i n g e r [33] d e m o n s t r a t e d that contraction of the frog h e a r t r a p i d l y ceased d u r i n g calciumfree perfusion at r o o m t e m p e r a t u r e , a n d was restored by the a d d i t i o n of calcium. Z i m m e r m a n a n d Hfilsmann [42] r e p o r t e d that in rat heart, restoration of a n o r m a l calcium concentration in the perfusate after a short period of perfusion w i t h o u t calcium at 37~ resulted in destruction of the m y o c a r d i a l cells (calcium p a r a d o x ) . T h e calcium p a r a d o x is characterized by an excessive influx of calcium into the m y o c a r d i a l cells [1], exhaustion of tissue highenergy phosphates [4, 7], massive release o f enzymes [19, 42], a n d extensive ultrastructural d a m a g e including severe c o n t r a c t u r e of the myofilaments and swelling o f the mitoc h o n d r i a with formation of electron-dense particles [27, 41]. W h e n the t e m p e r a t u r e d u r i n g the calcium-free period is reduced, the calcium p a r a d o x is less evident [3, 5, 20]. T h e p a r a d o x has been d e m o n s t r a t e d in rat, rabbit,
guinea-pig, mouse, dog and frog h e a r t [18, 35, 41], and also in h u m a n m y o c a r d i a l tissue [26]. D u r i n g calcium-free perfusion a separation between the surface coat and the external l a m i n a of the glycocalyx and a change in orientation of the i n t r a m e m b r a n e particles on the P-face of the s a r c o l e m m a have been observed [2, 12, 13]. A close correlation between this s e p a r a t i o n of the layers of the glycocalyx d u r i n g calcium depletion and the occurrence of the calcium p a r a d o x has been found [11]. In other studies, however, the role of the glycocalyx as the m a i n barrier to calcium entry into the cell has been questioned [28, 29]. Recently, a decrease in N a + / K + - A T P a s e activity of the s a r c o l e m m a a n d calcium p u m p activity of the sarcoplasmic reticulum has been observed in calcium depleted hearts [23, 24]. This would result in an i m p a i r e d a b i l i t y of the myocytes to remove calcium from the cytosol d u r i n g the reperfusion phase, thus c o n t r i b u t i n g to the net gain
* Address for reprint requests: Dr A. J. Verkleij, Institute of Molecular Biology, State University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands. 0022-2828/85/030265 +09 $03.00/0
9 1985 Academic Press Inc. (London) Limited
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in tissue calcium observed during the calcium paradox. The calcium paradox damage itself, which follows the massive influx of calcium into the myocardial cells, may be a result of energy dependent trans-membrane fluxes of calcium and a concomitant release of protons [17, 34]. It has also been proposed that calcium depletion causes loss of integrity of intercalated disc junctions, making the heart susceptible to physical stress-induced enzyme release [14, 15]. We have studied the ultrastructure of the sarcolemma of the rabbit heart during the calcium paradox by using fast freezing devices. This method allowed examination of the tissue without prior chemical fixation and cryoprotection, thus avoiding the risk of introducing artefacts. In addition to this ultrafast freezing method, we have used thin-section and conventional freeze-fracture electron microscopy. O u r findings show that the calcium paradox is accompanied with severe damage of the sarcolemma, including vesiculation and particle aggregation.
Materials
and Methods
Perfusion technique New Zealand white rabbits were anesthetized with H y p n o r m (fluanison/fentanyl) and thiopental. The animals were heparinized, the hearts were excised and subsequently perfused at 37~ by the Langendorff technique at a constant pressure of 6.4 kPa (65 cm HzO ). The standard perfusate ( + C a ) had the following composition (mmol/1): NaCl, 124.0; KC1, 4.7; MgC12, 1.0; N a H C O 3 , 24.0; Na2HPO4, 0.5; CaC1/, 1.3; glucose, 11.0. During calcium-free perfusion ( - Ca) calcium was omitted from the standard perfusate and no correction was made for the small change in osmolarity. The perfusion fluids were equilibrated with 95% 0 2 / 5 % CO 2 and the resulting p H was 7.4 at 37~ The following types of experiments were performed: (1) 60 min + C a (3 hearts); (2) 45 rain + C a , 15 rain - C a (3 hearts) ; (3) 40 rain + C a , 15 rain - C a , 5 min + C a (3 hearts); (4) 15min + C a , 15min - C a , 3 0 m i n + C a (3 hearts). All experiments, therefore, had a duration of 60 rain.
Electron microscopy At the end of the experiments, biopsy specimens of the left ventricle free wall were excised and cut into 1 m m 3 cubes. These were immersion-fixed for at least 2 h in glutaraldehyde (2.5%)/tannic acid (2%), which was added to the perfusate that was used during the last step of the experiment.
Thin-section electron microscopy After the first fixation the samples were washed in a 0.1 tool/1 phosphate buffer (pH 7.1) and post-fixed for 30 rain in a combination of 1% OsO 4 plus 1.5% K4Fe(CN)6. The samples were stained en bloc with 1% uranyl acetate in 70% acetone, dehydrated with the use of acetone and embedded in araldite resin. The blocks were sectioned on a Reichert O m U / u l t r a m i c r o t o m e with a diamond knife. The thin sections were mounted on copper grids, stained with uranyl acetate and lead citrate and examined in a Philips 301 or Philips 201 electron microscope.
Freeze-fracture electron microscopy conventional method After the glutaraldehyde/tannic acid fixation the samples were washed in the perfusate that was used during the last step of the experiment and placed in this perfusate containing 30% glycerol. Gold alloy hats containing tissue were frozen in a mixture of liquid and solid nitrogen. The fracturing and shadowing were performed in a Balzers BAF freeze-etch unit. Fracturing was done at a vacuum of 5 x 10 -v torr; specimen-table temperature was - 1 2 0 ~ and knife-temperature was -- 196~ Shadowing was done with a Balzers Control Unit E V M 052A. Thirty ~ngstroms of platinum-carbon was deposited (45 ~ angle), followed by t00 A of carbon (90 ~ angle).
Freeze-fracture electron microscopy --ultrafast method A one-sided propane jet was used for the ultrafast cryofixation of the specimens [22]. This method excluded ultrastructural changes induced by chemical fixation and cryoprotectants., Fracturing and shadowing were done in the same way as described above. The replicas obtained from both techniques were cleaned in a sodium hypochlorite solution and distilled water before mounting on copper
Sarcolemmal Disruption During Calcium Paradox
grids, and were examined in a Philips 301 or Philips 201 electron microscope. Results
267
Figures 3 and 4(b), resulted in the formation of particle-free regions of pure lipid. In contrast with the sarcolemma, the vesicles did not display many IMPs [Fig. 4(a)], indicating that the vesicles contained few intrinsic proteins. The aggregation of IMPs has also been observed by using conventional freezefracture electron microscopy [2, 13].
After 60 min of control perfusion the sarcolemma had an intact glycocalyx, which retained normal adherence to the bilayer [Fig. l(a)]. After 15 min calcium-free perfusion the glycocalyx exhibited lifting from the bilayer [Fig. l(b)], in accordance with Discussion other reports [12, 13, 19]. The bilayer itself remained continuous. The results of the present study show that After reperfusion with calcium-containing reperfusion of isolated rabbit hearts with solution the ultrastructural changes charac- calcium-containing solution, after a period of teristic of the calcium paradox were observed calcium-free perfusion, resulted in severe [2, 3, 7, 18-20, 27, 34, 41], such as contracture aggregation of intramembrane particles of the myofilaments and swelling of mitochon- (IMPs), as was demonstrated with an ultradria (result not shown). In addition to these fast freezing method before fracturing. This changes we observed alterations at the sarco- method excluded ultrastructural changes lemmal level. After 5 min reperfusion the lipid induced by chemical fixation and cryobilayer was no longer continuous and had protection. Reperfusion gave also rise to disformed vesicles. Some of these vesicles had a ruption of the sarcolemma and formation of multilamellar appearance [Fig. l(c), (e)]. unilamellar and multilamellar vesicles, as was After 30 min reperfusion the continuity of the demonstrated by thin-section and freezebilayer was completely lost and m a n y vesicles fracture electron microscopy. The vesicles had a smooth surface or, when their dimensions were observed [Fig. 1 (d)]. Both methods of freeze-fracturing con- were larger, contained few IMPs. The particle firmed the sarcolemmal disruption and density, however, was in all cases much vesiculation upon calcium repletion that were smaller than that of the sarcolemma, indicatobserved by using thin section electron ing that the vesicles contained few intrinsic microscopy. The replicas of the control per- proteins. fused hearts did not exhibit vesiculation of the Regarding the aggregation of the IMPs, it sarcolemma (result not shown). Figure 2 has been demonstrated that a redistribution of shows three replicas obtained by the conven- IMPs can be induced by proteases [6] and tional method of freeze-fracturing. After 15 phospholipases [_25, 39], and by changes of min calcium-free perfusion there was no temperature [16, 38], p H [10] and concentravesiculation of the sarcolemma [Fig. 2(a)], tion of divalent cations [10, 16]. Aggregation whereas after 5 and 30 min reperfusion with of IMPs as a result of protease activity only calcium-containing solution severe vesicu- occurs after long incubation times [6]. Phoslation was observed [Figs. 2(b), (c)]. A pholipases, which may be mobilized during number of vesicles had a multilamellar the calcium-free period because of an increased fluidity of the lipid bilayer, are actiappearance. The replicas of Figures 3 and 4 were vated during the reperfusion phase [21]. It is obtained by the ultrafast method of freeze- unlikely, however, that the subsequent formafracturing. By using this method, ultrastruc- tion of lysophospholipids in the bilayer may tural changes induced by chemical fixation account for the explosive character of the and cryoprotection were excluded. Severe calcium paradox damage. vesiculation of the sarcolemma and aggreAlternatively, aggregation of IMPs can be gation of intramembrane particles (IMPs) induced by calcium ions and protons (see [38] were observed after 5 and 30 min of calcium for a review). It has been demonstrated that repletion [Figs 3 and 4(a)]. This aggregation reperfusion of myocardial tissue with a of IMPs, which is clearly demonstrated in calcium-containing solution results in a net
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F I G U R E 1. Thin-section electron micrographs of isolated perfused rabbit hearts. (a) After 60 min control perfusion. The sarcolemma has an intact glycocalyx with normal adherence to the bilayer, x 60 000. (b) After 45 min control perfusion and 15 rain calcium-free perfusion. The glycocalyx exhibits lifting from the bilayer (arrows). x 60000. (c) After 40 min control perfusion, 15 min calcium-free perfusion, and 5 min of reperfusion. The bilayer is not continuous anymore and has formed vesicles, x 60 000. (d) After 15 min control perfusion, 15 min calcium-free perfusion, and 30 min ofreperfusion. The continuity of the bilayer is completely lost. Many vesicles can be observed, x 60 000. (e) Higher magnification of (c). Thismicrograph shows multilamellar vesicles (arrows). x 9 8 0 0 0 .
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F I G U R E 2. Freeze-fracture electron micrographs (conventional method) of isolated perfused rabbit hearts. (a) After 45 rain control perfusion and 15 min calcium-free perfusion. There is no vesiculation of the sarcolemma. S = sarcolemma; C = cytoplasm; M = mitochondrion, x60000. (b) After 15 min of control perfusion, 15 min calcium-free perfusion, and 30 rain reperfusion. Formation of sarcolemmal vesicles. A number of vesicles has a multilamellar appearance (arrow). C = cytoplasm, x 60 000. (c) After 40 rain control perfusion, 15 min calcium-free perfusion, and 5 min reperfusion. Formation of sarcolemmal vesicles (arrows). S = sarcolemma; C = cytoplasm. x 60 000.
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FIGURE 3. Freeze-fracture electron micrograph (ultrafast method) of isolated rabbit heart after 40 min control perfusion, 15 min calcium-free perfusion, and 5 min reperfusion. Notice severe aggregation of IMPs (arrows) and particle-freevesicles(arrowheads). x 52 500.
gain of tissue calcium [1], an extremely rapid breakdown of ATP [4, 7] by activation of the various ATPases, such as the myosin ATPase and the ATPase of the sarcoplasmic reticulum [17], and the formation ofintramitochondrial electron-dense particles [20, 40, 41]. Breakdown of ATP and mitochondrial calcium accumulation are accompanied by a release of protons [17, 34] and may lead to cytoplasmic acidification [17]. Under these circumstances redistribution of IMPs may be induced by interaction of calcium ions with negatively charged phospholipids (e.g. phosphatidylserine, PS), which leads to lateral phase separation of solid (PS) and liquid crystalline (phosphatidylcholine, PC and phosphatidylethanolamine, PE) lipids [9, 31]. PC, PE and PS comprise 39.2, 29.8 and 5.6% of the phospholipids of the rabbit sarcolemma, respectively [32]. This phase separation of phospholipids may be further
stimulated by lowering of the pH [36, 37]. Moreover, both calcium ions and protons can neutralize the negative charge of the intrinsic membrane proteins, which may also lead to the observed aggregation of IMPs [10]. Interaction of calcium ions with PS and a subsequent lateral phase separation of lipids may be also responsible for the extrusion of unilamellar and multilamellar vesicles, since it has been shown that PE has strongly fusogenic properties (see [8] for a review). PE and PS are likely localized in the cytoplasmic monolayer of the lipid bilayer [30, 39]. It has been demonstrated that a calcium concentration of approximately 0.2 mmol/1, in the presence of cytosolic levels of magnesium ions, induces fusion events in PE-PS-cholesterol systems [8]. Thus the formation of unilamellar and multilamellar sarcolemmal vesicles may be a result of the fusogenic property of PE. In summary, there is considerable support
Sarcolemmal Disruption During Calcium Paradox
27I
F I G U R E 4. Freeze-fracture electron micrographs (uhrafast method) of isolated rabbit hearts after 15 min control perfusion, 15 rain calcium-free perfusion, and 30 min reperfusion. (a) Notice severe vesiculation of the sarcolemma and aggregation of IMPs. The arrow indicates a multilamellar vesicle, x 34 000. (b) Notice aggregation of IMPs (arrows). x 50 000.
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for t h e h y p o t h e s i s t h a t t h e u n c o n t r o l l e d i n f l u x o f c a l c i u m i n t o t h e cells d u r i n g t h e p h a s e o f calcium repletion and the concomitant release o f p r o t o n s l e a d to t h e o b s e r v e d a g g r e g a t i o n o f I M P s a n d e x t r u s i o n o f l i p i d - e n r i c h e d vesicles. I t is p o s s i b l e t h a t these c h a n g e s p r e c e d e o r a r e c o i n c i d e n t w i t h t h e m a s s i v e release of cell c o n s t i t u e n t s , w h i c h is o n e o f t h e c h a r a c t e r i s t i c s o f the calcium paradox. However, the present results do not exclude that the sarcolemmal
d i s r u p t i o n is a n o n - s p e c i f i c effect o f cell n e c rosis. A d d i t i o n a l e x p e r i m e n t s a r e n e e d e d to establish the precise sequence of events that l e a d to s a r c o l e m m a l d i s r u p t i o n a n d to t h e release o f cell c o m p o n e n t s .
Acknowledgement T h e a u t h o r s a r e g r a t e f u l to M r D. d e H o e s for t e c h n i c a l a s s i s t a n c e , a n d to M i s s W . v a n E i j s d e n for s e c r e t a r i a l assistance.
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Sarcolemmal
Disruption During the Calcium Paradox
j. A. Post, P. F. E. M. Nievelstein, J. Leunissen-Bijvelt, A. J. Verkleij* and T. J. C. Rulgrok~ Department of Molecular Cell Biology, and *Institute of Molecular Biology, State University of Utrecht, Utrecht, The Netherlands and t Department of Cardiology, University Hospital, Utrecht, The Netherlands (Received 1 February 1984, accepted in revisedform 21 August 1984) j. A. POST,P. F. E. M. NIEVELSTEIN,J. LEUNISSEN-BIJVELT,A.J.
VERKLEIJ AND T.J.C. RUIGROK.Sarcolemmal Disruption During the Calcimn Paradox. Journalof Molecularand CellularCardiology(1985) 17, 265 273. Reperfusion of an isolated heart with calcium-containing solution after a short period of calcium-free perfusion may result in irreversible cell damage (calcium paradox). The ultrastructure of the sarcolemma of the rabbit heart during the calcium paradox was studied by using fast freezing devices. This method excluded ultrastructural changes induced by chemical fixation and cryoprotection. In addition, thin-section and conventional freezefracture electron microscopy were used. During reperfusion with calcium-containing solution disruption of the sarcolemma was observed, which was attended with formation of unilamellar and multilamellar vesicles and aggregation of intramembrane particles. These ultrastructural changes are explained in terms of calcium- and proton-induced lateral phase separation and fusion processesin the lipid bilayer of the sarcolemma.
KEY WORDS: Calcium paradox; Rabbit hearts; Freeze-fracture electron microscopy; Fast freezing devices; Sarcolemma ; Phospholipids ; Vesiculation; Intramembrane particles.
Introduction R i n g e r [33] d e m o n s t r a t e d that contraction of the frog h e a r t r a p i d l y ceased d u r i n g calciumfree perfusion at r o o m t e m p e r a t u r e , a n d was restored by the a d d i t i o n of calcium. Z i m m e r m a n a n d Hfilsmann [42] r e p o r t e d that in rat heart, restoration of a n o r m a l calcium concentration in the perfusate after a short period of perfusion w i t h o u t calcium at 37~ resulted in destruction of the m y o c a r d i a l cells (calcium p a r a d o x ) . T h e calcium p a r a d o x is characterized by an excessive influx of calcium into the m y o c a r d i a l cells [1], exhaustion of tissue highenergy phosphates [4, 7], massive release o f enzymes [19, 42], a n d extensive ultrastructural d a m a g e including severe c o n t r a c t u r e of the myofilaments and swelling o f the mitoc h o n d r i a with formation of electron-dense particles [27, 41]. W h e n the t e m p e r a t u r e d u r i n g the calcium-free period is reduced, the calcium p a r a d o x is less evident [3, 5, 20]. T h e p a r a d o x has been d e m o n s t r a t e d in rat, rabbit,
guinea-pig, mouse, dog and frog h e a r t [18, 35, 41], and also in h u m a n m y o c a r d i a l tissue [26]. D u r i n g calcium-free perfusion a separation between the surface coat and the external l a m i n a of the glycocalyx and a change in orientation of the i n t r a m e m b r a n e particles on the P-face of the s a r c o l e m m a have been observed [2, 12, 13]. A close correlation between this s e p a r a t i o n of the layers of the glycocalyx d u r i n g calcium depletion and the occurrence of the calcium p a r a d o x has been found [11]. In other studies, however, the role of the glycocalyx as the m a i n barrier to calcium entry into the cell has been questioned [28, 29]. Recently, a decrease in N a + / K + - A T P a s e activity of the s a r c o l e m m a a n d calcium p u m p activity of the sarcoplasmic reticulum has been observed in calcium depleted hearts [23, 24]. This would result in an i m p a i r e d a b i l i t y of the myocytes to remove calcium from the cytosol d u r i n g the reperfusion phase, thus c o n t r i b u t i n g to the net gain
* Address for reprint requests: Dr A. J. Verkleij, Institute of Molecular Biology, State University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands. 0022-2828/85/030265 +09 $03.00/0
9 1985 Academic Press Inc. (London) Limited
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in tissue calcium observed during the calcium paradox. The calcium paradox damage itself, which follows the massive influx of calcium into the myocardial cells, may be a result of energy dependent trans-membrane fluxes of calcium and a concomitant release of protons [17, 34]. It has also been proposed that calcium depletion causes loss of integrity of intercalated disc junctions, making the heart susceptible to physical stress-induced enzyme release [14, 15]. We have studied the ultrastructure of the sarcolemma of the rabbit heart during the calcium paradox by using fast freezing devices. This method allowed examination of the tissue without prior chemical fixation and cryoprotection, thus avoiding the risk of introducing artefacts. In addition to this ultrafast freezing method, we have used thin-section and conventional freeze-fracture electron microscopy. O u r findings show that the calcium paradox is accompanied with severe damage of the sarcolemma, including vesiculation and particle aggregation.
Materials
and Methods
Perfusion technique New Zealand white rabbits were anesthetized with H y p n o r m (fluanison/fentanyl) and thiopental. The animals were heparinized, the hearts were excised and subsequently perfused at 37~ by the Langendorff technique at a constant pressure of 6.4 kPa (65 cm HzO ). The standard perfusate ( + C a ) had the following composition (mmol/1): NaCl, 124.0; KC1, 4.7; MgC12, 1.0; N a H C O 3 , 24.0; Na2HPO4, 0.5; CaC1/, 1.3; glucose, 11.0. During calcium-free perfusion ( - Ca) calcium was omitted from the standard perfusate and no correction was made for the small change in osmolarity. The perfusion fluids were equilibrated with 95% 0 2 / 5 % CO 2 and the resulting p H was 7.4 at 37~ The following types of experiments were performed: (1) 60 min + C a (3 hearts); (2) 45 rain + C a , 15 rain - C a (3 hearts) ; (3) 40 rain + C a , 15 rain - C a , 5 min + C a (3 hearts); (4) 15min + C a , 15min - C a , 3 0 m i n + C a (3 hearts). All experiments, therefore, had a duration of 60 rain.
Electron microscopy At the end of the experiments, biopsy specimens of the left ventricle free wall were excised and cut into 1 m m 3 cubes. These were immersion-fixed for at least 2 h in glutaraldehyde (2.5%)/tannic acid (2%), which was added to the perfusate that was used during the last step of the experiment.
Thin-section electron microscopy After the first fixation the samples were washed in a 0.1 tool/1 phosphate buffer (pH 7.1) and post-fixed for 30 rain in a combination of 1% OsO 4 plus 1.5% K4Fe(CN)6. The samples were stained en bloc with 1% uranyl acetate in 70% acetone, dehydrated with the use of acetone and embedded in araldite resin. The blocks were sectioned on a Reichert O m U / u l t r a m i c r o t o m e with a diamond knife. The thin sections were mounted on copper grids, stained with uranyl acetate and lead citrate and examined in a Philips 301 or Philips 201 electron microscope.
Freeze-fracture electron microscopy conventional method After the glutaraldehyde/tannic acid fixation the samples were washed in the perfusate that was used during the last step of the experiment and placed in this perfusate containing 30% glycerol. Gold alloy hats containing tissue were frozen in a mixture of liquid and solid nitrogen. The fracturing and shadowing were performed in a Balzers BAF freeze-etch unit. Fracturing was done at a vacuum of 5 x 10 -v torr; specimen-table temperature was - 1 2 0 ~ and knife-temperature was -- 196~ Shadowing was done with a Balzers Control Unit E V M 052A. Thirty ~ngstroms of platinum-carbon was deposited (45 ~ angle), followed by t00 A of carbon (90 ~ angle).
Freeze-fracture electron microscopy --ultrafast method A one-sided propane jet was used for the ultrafast cryofixation of the specimens [22]. This method excluded ultrastructural changes induced by chemical fixation and cryoprotectants., Fracturing and shadowing were done in the same way as described above. The replicas obtained from both techniques were cleaned in a sodium hypochlorite solution and distilled water before mounting on copper
Sarcolemmal Disruption During Calcium Paradox
grids, and were examined in a Philips 301 or Philips 201 electron microscope. Results
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Figures 3 and 4(b), resulted in the formation of particle-free regions of pure lipid. In contrast with the sarcolemma, the vesicles did not display many IMPs [Fig. 4(a)], indicating that the vesicles contained few intrinsic proteins. The aggregation of IMPs has also been observed by using conventional freezefracture electron microscopy [2, 13].
After 60 min of control perfusion the sarcolemma had an intact glycocalyx, which retained normal adherence to the bilayer [Fig. l(a)]. After 15 min calcium-free perfusion the glycocalyx exhibited lifting from the bilayer [Fig. l(b)], in accordance with Discussion other reports [12, 13, 19]. The bilayer itself remained continuous. The results of the present study show that After reperfusion with calcium-containing reperfusion of isolated rabbit hearts with solution the ultrastructural changes charac- calcium-containing solution, after a period of teristic of the calcium paradox were observed calcium-free perfusion, resulted in severe [2, 3, 7, 18-20, 27, 34, 41], such as contracture aggregation of intramembrane particles of the myofilaments and swelling of mitochon- (IMPs), as was demonstrated with an ultradria (result not shown). In addition to these fast freezing method before fracturing. This changes we observed alterations at the sarco- method excluded ultrastructural changes lemmal level. After 5 min reperfusion the lipid induced by chemical fixation and cryobilayer was no longer continuous and had protection. Reperfusion gave also rise to disformed vesicles. Some of these vesicles had a ruption of the sarcolemma and formation of multilamellar appearance [Fig. l(c), (e)]. unilamellar and multilamellar vesicles, as was After 30 min reperfusion the continuity of the demonstrated by thin-section and freezebilayer was completely lost and m a n y vesicles fracture electron microscopy. The vesicles had a smooth surface or, when their dimensions were observed [Fig. 1 (d)]. Both methods of freeze-fracturing con- were larger, contained few IMPs. The particle firmed the sarcolemmal disruption and density, however, was in all cases much vesiculation upon calcium repletion that were smaller than that of the sarcolemma, indicatobserved by using thin section electron ing that the vesicles contained few intrinsic microscopy. The replicas of the control per- proteins. fused hearts did not exhibit vesiculation of the Regarding the aggregation of the IMPs, it sarcolemma (result not shown). Figure 2 has been demonstrated that a redistribution of shows three replicas obtained by the conven- IMPs can be induced by proteases [6] and tional method of freeze-fracturing. After 15 phospholipases [_25, 39], and by changes of min calcium-free perfusion there was no temperature [16, 38], p H [10] and concentravesiculation of the sarcolemma [Fig. 2(a)], tion of divalent cations [10, 16]. Aggregation whereas after 5 and 30 min reperfusion with of IMPs as a result of protease activity only calcium-containing solution severe vesicu- occurs after long incubation times [6]. Phoslation was observed [Figs. 2(b), (c)]. A pholipases, which may be mobilized during number of vesicles had a multilamellar the calcium-free period because of an increased fluidity of the lipid bilayer, are actiappearance. The replicas of Figures 3 and 4 were vated during the reperfusion phase [21]. It is obtained by the ultrafast method of freeze- unlikely, however, that the subsequent formafracturing. By using this method, ultrastruc- tion of lysophospholipids in the bilayer may tural changes induced by chemical fixation account for the explosive character of the and cryoprotection were excluded. Severe calcium paradox damage. vesiculation of the sarcolemma and aggreAlternatively, aggregation of IMPs can be gation of intramembrane particles (IMPs) induced by calcium ions and protons (see [38] were observed after 5 and 30 min of calcium for a review). It has been demonstrated that repletion [Figs 3 and 4(a)]. This aggregation reperfusion of myocardial tissue with a of IMPs, which is clearly demonstrated in calcium-containing solution results in a net
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F I G U R E 1. Thin-section electron micrographs of isolated perfused rabbit hearts. (a) After 60 min control perfusion. The sarcolemma has an intact glycocalyx with normal adherence to the bilayer, x 60 000. (b) After 45 min control perfusion and 15 rain calcium-free perfusion. The glycocalyx exhibits lifting from the bilayer (arrows). x 60000. (c) After 40 min control perfusion, 15 min calcium-free perfusion, and 5 min of reperfusion. The bilayer is not continuous anymore and has formed vesicles, x 60 000. (d) After 15 min control perfusion, 15 min calcium-free perfusion, and 30 min ofreperfusion. The continuity of the bilayer is completely lost. Many vesicles can be observed, x 60 000. (e) Higher magnification of (c). Thismicrograph shows multilamellar vesicles (arrows). x 9 8 0 0 0 .
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F I G U R E 2. Freeze-fracture electron micrographs (conventional method) of isolated perfused rabbit hearts. (a) After 45 rain control perfusion and 15 min calcium-free perfusion. There is no vesiculation of the sarcolemma. S = sarcolemma; C = cytoplasm; M = mitochondrion, x60000. (b) After 15 min of control perfusion, 15 min calcium-free perfusion, and 30 rain reperfusion. Formation of sarcolemmal vesicles. A number of vesicles has a multilamellar appearance (arrow). C = cytoplasm, x 60 000. (c) After 40 rain control perfusion, 15 min calcium-free perfusion, and 5 min reperfusion. Formation of sarcolemmal vesicles (arrows). S = sarcolemma; C = cytoplasm. x 60 000.
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FIGURE 3. Freeze-fracture electron micrograph (ultrafast method) of isolated rabbit heart after 40 min control perfusion, 15 min calcium-free perfusion, and 5 min reperfusion. Notice severe aggregation of IMPs (arrows) and particle-freevesicles(arrowheads). x 52 500.
gain of tissue calcium [1], an extremely rapid breakdown of ATP [4, 7] by activation of the various ATPases, such as the myosin ATPase and the ATPase of the sarcoplasmic reticulum [17], and the formation ofintramitochondrial electron-dense particles [20, 40, 41]. Breakdown of ATP and mitochondrial calcium accumulation are accompanied by a release of protons [17, 34] and may lead to cytoplasmic acidification [17]. Under these circumstances redistribution of IMPs may be induced by interaction of calcium ions with negatively charged phospholipids (e.g. phosphatidylserine, PS), which leads to lateral phase separation of solid (PS) and liquid crystalline (phosphatidylcholine, PC and phosphatidylethanolamine, PE) lipids [9, 31]. PC, PE and PS comprise 39.2, 29.8 and 5.6% of the phospholipids of the rabbit sarcolemma, respectively [32]. This phase separation of phospholipids may be further
stimulated by lowering of the pH [36, 37]. Moreover, both calcium ions and protons can neutralize the negative charge of the intrinsic membrane proteins, which may also lead to the observed aggregation of IMPs [10]. Interaction of calcium ions with PS and a subsequent lateral phase separation of lipids may be also responsible for the extrusion of unilamellar and multilamellar vesicles, since it has been shown that PE has strongly fusogenic properties (see [8] for a review). PE and PS are likely localized in the cytoplasmic monolayer of the lipid bilayer [30, 39]. It has been demonstrated that a calcium concentration of approximately 0.2 mmol/1, in the presence of cytosolic levels of magnesium ions, induces fusion events in PE-PS-cholesterol systems [8]. Thus the formation of unilamellar and multilamellar sarcolemmal vesicles may be a result of the fusogenic property of PE. In summary, there is considerable support
Sarcolemmal Disruption During Calcium Paradox
27I
F I G U R E 4. Freeze-fracture electron micrographs (uhrafast method) of isolated rabbit hearts after 15 min control perfusion, 15 rain calcium-free perfusion, and 30 min reperfusion. (a) Notice severe vesiculation of the sarcolemma and aggregation of IMPs. The arrow indicates a multilamellar vesicle, x 34 000. (b) Notice aggregation of IMPs (arrows). x 50 000.
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for t h e h y p o t h e s i s t h a t t h e u n c o n t r o l l e d i n f l u x o f c a l c i u m i n t o t h e cells d u r i n g t h e p h a s e o f calcium repletion and the concomitant release o f p r o t o n s l e a d to t h e o b s e r v e d a g g r e g a t i o n o f I M P s a n d e x t r u s i o n o f l i p i d - e n r i c h e d vesicles. I t is p o s s i b l e t h a t these c h a n g e s p r e c e d e o r a r e c o i n c i d e n t w i t h t h e m a s s i v e release of cell c o n s t i t u e n t s , w h i c h is o n e o f t h e c h a r a c t e r i s t i c s o f the calcium paradox. However, the present results do not exclude that the sarcolemmal
d i s r u p t i o n is a n o n - s p e c i f i c effect o f cell n e c rosis. A d d i t i o n a l e x p e r i m e n t s a r e n e e d e d to establish the precise sequence of events that l e a d to s a r c o l e m m a l d i s r u p t i o n a n d to t h e release o f cell c o m p o n e n t s .
Acknowledgement T h e a u t h o r s a r e g r a t e f u l to M r D. d e H o e s for t e c h n i c a l a s s i s t a n c e , a n d to M i s s W . v a n E i j s d e n for s e c r e t a r i a l assistance.
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