Role of calcium in triggering rapid ultrastructurai damage in muscle:

a study with chemically skinned fibres

C. J. DUNCAN

Department of Zoology, University of Liverpool, PO Box 147, Liverpool L69 3BX, UK

Summary

Agents (A23187, caffeine) believed to raise [Ca]jin vertebrate cardiac and skeletal muscles causerapid and characteristic subcellular damage invitro and in vivo. By using saponin-skinned am-phibian pectoris cutaneous muscle and Ca-EGTA-buffered solutions it is shown that low[Ca] consistently triggers the same rapid(2-20 min), ultrastructurai damage.

Electron micrographs reveal a close similaritybetween the damaged intact and skinned prep-arations, namely loss of myofilament organiz-ation, specific Z-line damage, dissolution andhypercontraction bands, characteristic mitochon-drial swelling and division. Where both actin andmyosin filaments 'were lost, an underlying cyto-skeletal network frequently remained, still at-tached to the Z-line framework.

Ca was effective in skinned preparations from5xlO~7M to 8xlO~6M, within the concentrationrange experienced by a contracting muscle.

Damage was [Ca]- and time-dependent and it issuggested that it is probably the active movementof Ca ions across key membrane sites that iscritical in triggering damage of the myofilamentapparatus. Strontium can substitute for Ca athigher concentrations. The action of saponinsuggests that the chemically skinned cell is par-tially activated. Ca-triggering can be bypassedexperimentally by membrane-active agents or bysulphydryl agents.

Ruthenium Red and trifluoperazine indirectlycause damage in the intact cell by raising [Ca]j.

Studies 'with saponin-skinned cells and pro-tease inhibitors show that changes in pHj, loss ofATP, Ca-activated neutral protease, or release oflysosomal enzymes (cathepsins B, D, L or H), arenot involved in characteristic rapid myofilamentdamage.

Key words: calcium, muscle damage, lysosomes, calcium-activated neutral protease.

Introduction

There is considerable current interest in the triggeringand regulation of cellular breakdown and death in avariety of tissues including brain, liver, muscle andkidney as well as in insects and amphibians undergoingmetamorphosis. A range of different factors have beenimplicated, e.g. lysosomal and non-lysosomal pro-teases, energy-dependent and energy-independent pro-teases, cellular supply of high-energy phosphates,thyroid hormones, impaired mitochondrial function-ing, prostaglandins, phospholipase A2 activity, endo-toxaemia, vitamin E deficiency, oxygen radicals andtrauma.

The suggestion that an alteration in intracellular Cahomeostasis was an important step in the cascade ofevents that culminate in cellular damage (Duncan,

Journal of Cell Science 87, 581-594 (1987)Printed in Great Britain © The Company of Biologists Limited 1987

1978; Schanne et al. 1979; Katz & Reuter, 1979;Farber, 1981; Trump et al. 1981; Ishiura, 1981;Nayler, 1983) provided a unifying hypothesis withimportant medical implications for many degradativeconditions. However, in opposition to this hypothesis,the following conclusions have recently been advanced:"chemical-induced hepatic cell death is not caused byan increase in total cellular Ca2+ resulting from aninflux of extracellular Ca2+" (Fariss & Reed, 1985),and "We report here that free Ca in metabolically-poisoned myocytes is remarkably stable and that severeinjury to the cell occurs before the free Ca concen-tration rises above 1 to 3xlO~7M, hence cell damageseems to be a cause, not a consequence of a rise in freeCa" (Cobbold & Bourne, 1984).

It is well known that subcellular damage in bothskeletal and cardiac muscle can be produced rapidly,

Calcium and muscle damage 581

i.e. with a time-course of minutes; e.g. the ischaemicmammalian heart and skeletal muscle during malignanthyperthermia. These clinical events can be replicatedexperimentally not only in vivo, as in studies of thecalcium paradox and ischaemia of cardiac muscle, butalso in vitro, as in ultrastructural studies of bothskeletal and cardiac muscle. Many of these examples ofrapid subcellular damage are triggered by agents thatappear to raise [Ca], in the muscle (both cardiac andskeletal) or to promote Ca fluxes: (1) the Ca paradox ofcardiac muscle (Grinwald & Nayler, 1981; Opie,1985); (2) the actions of the divalent cation ionophoreA23187 (Statham et al. 1976; Publicover et al. 1978)where ultrastructural damage has now been linked torises in [Ca], (Yoshimura et al. 1986); (3) the action ofDantrolene sodium in preventing the release of Cafrom the sarcoplasmic reticulum (SR) and protectingagainst malignant hyperthermia (Blanck & Gruener,1983); (4) the effect of the withdrawal of extracellularCa in protecting against protein efflux from isolatedmouse muscle when challenged with excessive contrac-tile activity in anoxia (Jones et al. 1984).

The conclusion from these studies is that abruptrises in intracellular Ca are able to trigger rapid andcharacteristic ultrastructural damage and that there is acommon degradative mechanism of cardiac and skeletalmuscle in both amphibians and mammals.

However, we have, as yet, no direct measurements ofintracellular [Ca] during the process of damage inskeletal muscle and thus the first objective of thepresent study was to develop a chemically skinnedskeletal muscle fibre preparation in which intracellularCa could be accurately controlled. It is shown that a[Ca] of 10~6M can consistently trigger rapid andcharacteristic damage.

Second, the skinned fibre preparation was used toassess the importance of proteolytic enzymes in cellulardamage. One hypothesis concerns the involvement ofCa-activated neutral proteases (CANP) and, alterna-tively, the Ca-activated damage process might involvelysosomal labilization and breakdown. Thus, DNP isone agent that is also known to cause release oflysosomal hydrolases only via mechanisms that operatein the intact cell (Martini, 1959), whilst an increase inlysosomal catheptic enzyme activity has been reportedin various muscular dystrophies (Pearson & Kar,1979). It is reported here that a variety of proteaseinhibitors failed to prevent rapid damage in the skinnedmuscle cell. Finally, other mechanisms have beensuggested concerning the triggering of damage inmuscle cells, such as a fall in pH; or a severe depletionof high-energy phosphates. The skinned-fibre prep-aration was used to adjust and control the intracellularenvironment and to test these hypotheses.

Materials and methods

Frogs

Rana pipiens were maintained in the laboratory at 23 °C andfed on blowfly larvae; R. temporaria were maintained at 9°C.Pectoris cutaneous muscles were isolated and pinned todental wax in ice-cold Ringer solution lacking Ca2+.

SkinningThe preparations were transferred to one of the two followingmedia of composition (mM): (1) sucrose (250), piperazine-iVVV'-bis[2-ethanesulphonic acid] (4), magnesium acetate(4), EGTA (0-5), ATP (Na salt) (3), brought to pH 7 1 withNaOH and KOH so that [K + ]=2-5 and [Na+] = 10.(2) Potassium glutamate (100), piperazine-jVr(V'-bis[2-eth-anesulphonic acid] (10), magnesium acetate (4), EGTA(0-5), ATP (Na salt) (3), pH7-l with KOH. Temperature,18CC. Saponin was added to the skinning solution immedi-ately before use and briskly stirred.

Exposure of skinned preparations to test solutions: skinnedpreparations were rinsed in ice-cold potassium glutamatesolution (medium (2)) and then transferred to fresh potass-ium glutamate medium (18CC). Samples of 1 M-CaCl2 (Ana-laR) or of SrCl2 were added to produce calculated fixed freedivalent cation concentrations in the EGTA buffer systems,using the appropriate pK values as given by Bjerrum et al.(1957). Protease inhibitors were dissolved in potassiumglutamate medium immediately before use; chymostatin wassolubilized in DMSO (dimethyl sulphoxide), pepstatin inethanol.

Unskinned preparationsCutaneous pectoris muscles were pinned out in Ringer'ssolution containing l-8mM-Ca and 5^gml~' A23187, whichwas initially solubilized in ethanol. Temperature, 20°C;pH7-l .

Electron microscopyFixation was by one of three methods: (1) Karnovsky fixativeat 20°C for 30 min, with transfer to fresh fixative for a further3-5 h, followed by two washes with 0-1 M-sodium cacodylate,pH7-2, each of 30 min. The tissue was then cut into smallerpieces and poatfixed in OSO4 for 2h at room temperature.The pieces of muscle were then cut into small blocks forwashing in cacodylate buffer (two changes). (2) Glutaralde-hyde (3 %) with a protocol as in method (1). (3) OsO4 (1 %)in venonal acetate buffer, pH 7-2 at 20°C for 3-5 h, followedby two washes in 0-l M-sodium cacodylate, pH7-2, each of30 min. All preparations were dehydrated through a gradedethanol series, embedded in Spurr's resin and sections werecut at 60— 90 nm and stained with uranyl acetate and leadcitrate.

Assessment of ultrastructural changesAt least three separate samples were taken from each muscleand the sections were scored blind on the electron micro-scope, by a colleague, for different types of damage; finally,the electron micrographs were again independently assessed.

582 C. J. Duncan

ReagentsSigma Chemical Co., Poole, Dorset: A23187, aprotinin(A1153 from bovine lung), pepstatin A, Ruthenium Red,caffeine. Peptide Institute Inc., Osaka, Japan: chymostatin,leupeptin.

Results

Exposure of intact fibres to A23187This divalent cation ionophore caused characteristicand progressive damage in the intact cutaneous pectorisof R. temporaria over 10-30 min exposure. Fixationwas with Karnovsky fixative. Initially, Z-line streamingwas detected but, after some 20 min, breakdown ofmyofibrils occurred and some were contracted inde-pendently with Z-lines clearly out of register. After20-30min damage was widespread and of two types:(1) dissolution and breakdown of the myofilaments, or(2) a form of hypercontraction with very blurred Z-lines. Exposure of intact fibres to 5 mM or 8 mM-caffeine for 30 min also caused the same characteristicultrastructural damage.

Preparation of skinned fibres

A total of 240 skinned cutaneous pectoris preparationswere used in the present study and it was apparent thatthere were differences between R. pipiens and R.temporaria as well as marked variability betweenpreparations (see below). Exposure to high saponinconcentrations alone could promote ultrastructuraldamage and its effects were augmented by treatmentsthat elevated intracellular [Ca]. Thus, the sucrosemedium containing 0-5mM-EGTA (rather than thepotassium glutamate) proved more satisfactory and thenormal concentrations of saponin used were: R. pipiens37 • 5 fig m\~l, 25min; R. temporaria oO^igmP1,30 min. Electron micrographs (Fig. 1) of skinned prep-arations maintained for 90 min revealed a normalultrastructure with clear myofilaments, sharp Z-lines inregister, contracted mitochondria and with the SR notswollen.

Fixation of skinned-fibre preparationsMajor changes resulting from damage could be readilydetected with transmitted light and X30 magnificationin skinned preparations of the thin cutaneous pectorismuscle. Fixation with glutaraldehyde-based fixativesproduced severe and visible damage within 60s ofapplication and hence subsequently all fixation waswith OsO4.

Action of Ca

Exposure of skinned fibres to potassium glutamatesolutions in which Ca was buffered at 8xlO~6Mconsistently produced damage, which was severewithin 10 min in R. pipiens preparations and within

25 min in R. temporaria, and was readily detectable atX30 magnification. Initially, this damage was seen asan area of contraction and abnormal folding of themuscle sheet, which gradually increased in area. Later,the contents in individual cells could be seen concen-trated at specific foci and surrounded by clear cyto-plasm.

Electron micrographs of Ca-damaged, skinned fibresrevealed characteristic patterns of damage and, as withthe intact cutaneous pectoris preparations, the resultswere invariably clear-cut. Whilst areas of undamagedmyofibrils were found in all but the severest examplesof damage, muscles in which the damage mechanismhad been activated were immediately recognizable.The different types of degradative effect are listed inapproximate order of severity in Table 1. Althoughindividual sarcomeres appear to respond differently tothe damage process (see individual myofibrils in Figs 3and 14), there was a distinction, as with the resultsobtained with intact fibres, between sections of themuscle where damage was accompanied by extremecontraction (categories 6 and 7, Table 1), where themyofilament apparatus was clearly damaged but com-plete destruction did not occur during the 30-minexposure period (Fig. 11), and where hypercontractiondid not occur and complete loss of actin and myosinfilaments was found (Figs 4, 5, 19). Fig. 14 illustrates

Table 1. Ultrastructural damage recorded inskinned fibres of amphibian pectoris cutaneous

muscles

Category

1

23

45

6

7

89

10

11

Appearance

Small areas with loss ofmyofilament organization andZ-hne damage; frequently nocontraction

Z-line slidingSeparation of fibrils, sometimes

accompanied by SR swellingSarcomeres relaxedSarcomeres contracted

Specific loss of actin filamentsProgressive damage to

myofilaments; eventually onlyZ-lines and cytoskeletalelements remain

Contraction of the fibril, withheavily blurred Z-line

Hypercontraction bands withamorphous material; only darkZ-lines distinguishable

Swollen vesiclesMitochondria swollen, damaged,

dividingDissolution of cytoplasmic

componentsNuclear damage; margination and

clumping of heterochromatin,imagination of the nuclearmembrane

Figs

2, 3

2 ,74 ,9

4, 56, 97, 817, 18, 19

10, 12, 13

11, 12, 14

9, 11, 14,11, 14, 15

5, 6, 13, 1

, 21

, 16

16, 16

4

Calcium and muscle damage 583

W44w!&

Figs 1-6. Ca-triggered damage in saponin-skinned pectoris cutaneous muscle cells; Bar, 1 Jim, except Fig. 4.Fig. 1. Undamaged area; 8xlO~6M-Ca, 25min; R. temporaria.Fig. 2. Early damage with Z-line sliding and disintegration; 5xlO~7M-Ca, 25min; R. pipiens.Fig. 3. Widespread, early damage, swollen vesicles; 8xlO~6M-Ca, 40min; R. temporaria.Fig. 4. Marked separation of relaxed myofibrils with dissolution of parts of the myofilament apparatus; 5xlO~5M-Ca,

28min; R. temporaria. Bar, 2jmi.Fig. 5. Relaxed, broken myofibrils with normal myofilament apparatus but surrounded by the remnants of the other

cytoplasmic components; 8xlO~6M-Ca plus 14jigml~' pepstatin, 25min; R. pipiens.Fig. 6. As Fig. 5, but with myofibrils contracted; 5xlO~7M-Ca, Zbmin; R. pipiens.

584 C. jf. Duncan

8

Figs 7, 8. Examples of saponin-skinned pectoris cutaneousfibres where the damage is concentrated on the actinfilaments, and is accompanied by marked Z-line sliding andby the extension of the sarcomeres. R. pipiens. Fig. 7,8xlO~6M-Ca plus 5 0 ^ g m r ' leupeptin, 25 min. Bar, 1/wn.

4Fig. 8, 5X1O"4M-Sr, 25 min.

hypercontraction and complete dissolution in the samefibril. All these different types of damage were found inboth species, but substantial differences could be foundin different areas of the same muscle sheet; thusdifferent categories of damage (together with appar-ently undamaged sections) could be found in onepreparation and in adjacent sections of the samemyofibril. Contraction-type damage (categories 6 and7) appeared to be associated with the contractionsobserved with transmitted light. It is evident that thepatterns of damage correspond with those caused byA23187 in intact fibres (Statham et al. 1976) whilst thehypercontraction bands (category 7; Figs 11, 12, 14),in particular, also compare closely to those found in

amphibian cardiac muscle (Rudge & Duncan, 1984a).We conclude that the rapid Ca-triggered damage ofskinned muscle fibres is identical with that found inintact fibres.

A series of experiments with both species showedthat the effect of Ca was both time- and concentration-dependent. With the R. pipiens preparations, ultra-structural changes were not detected with [Ca] =8xlO~8M and developed only slowly in some prep-arations at [Ca] = 10~7M. In most preparationsdamage was detectable by eye in less than 10 min at5xlO~7M-Ca and in all preparations in less than 5 minat 10~6M-Ca, and in less than 2 min at 8X 10~6M. TheR. temporaria preparation (summer frogs, skinnedwith fjOf/gml"1 saponin), however, was consistentlyless sensitive and damage was not evident in theelectron micrographs after 10 min exposure to8xlO~6M-Ca, but usually began after 18-20min.Twenty to 25 min were required with 10~6M andusually 30 min at 5xlO~7M. It is noteworthy that atlow [Ca] ( < 1 0 ~ 6 M ) the damage seen was more com-monly in categories 1-5 but, nevertheless, contraction-type damage (categories 6 and 7) was also recorded andthus the same damage response was found across theCa concentration range.

Cytoskeletal elements of the amphibian sarcomereSome preparations showing category 5 damage (Table1), where much of the contractile apparatus had beenlost, revealed apparent thin, cytoskeletal, longitudinalelements linking the Z-lines, which appeared to bemore resistant to the rapid degradative process than themyofilament apparatus. This characteristic effect wasfound in both intact (Fig. 20) and skinned(Figs 17-19, 21) amphibian muscle cells in which thedegradative mechanism had been switched on in avariety of different ways. The appearance of thesemuscle cells closely resembled those of frog prep-arations in which the actin and myosin had beenextracted with 0 ' 6 M - K I , SO revealing the connectin (ortitin/nebulin) network (Wang, 1982). This parallelelastic component (Maruyama et al. 1985) appearstherefore, to be more resistant to the rapid degradativeprocess than are the bulk myofibrillar proteins.

Effect of saponin

The sensitivity of some preparations to Ca and thevariability between different muscles suggested thatsaponin might directly activate the damage process aswell as permeabilize the sarcolemma. R. temporariamuscles were skinned for 30 min in a range of saponinconcentrations, namely 40, 50, 55, 60, 75, 100, 120 and150/igmP1, and Ca-induced damage was found todevelop more rapidly with much more severe effects at

Calcium and muscle damage 585

the higher concentrations. At 150;Ugml , 8 x 1 0 M- experiments, 150/zgml saponin directly causedCa damage could be detected in 90-120 s by transmit- damage in R. temporaria.ted light and, after 9min, electron micrographs re-vealed almost complete dissolution of wide areas; a Action of strontiumvery much more dramatic response than with skinning Sr-EGTA buffered solutions also triggered identicalwith 60^gml~' saponin in this species. In one series of patterns of damage in R. pipiens skinned preparations

586 C. jf. Duncan

(Fig. 8), although substantially higher concentrationsof free Sr were required. At 3xlO~6M-Sr (25 min)individual damaged sarcomeres were detected togetherwith some Z-line sliding; at 10~5M-Sr (after 45 min)the SR was additionally swollen in some areas; at5xlO~sM-Sr (25 min) substantial parts of the muscleremained undamaged but characteristic areas of myo-filament breakdown were evident (category 1, Table 1).Finally, with 5xlO~4M-Sr (25 min), clearly evidentcontractions and folding were seen under transmittedlight and electron micrographs showed a full range ofsevere damage, including areas of extensive hypercon-traction and mitochondrial multiplication (categories6-9).

Ruthenium Red

Exposure of the R. pipiens skinned muscle to the Ca-ATPase inhibitor Ruthenium Red (2, 10 or 15jUM,20 min) in the potassium glutamate-EGTA medium,with no added Ca, did not cause the characteristicdamage.

Effect ofpH

The skinned preparations were exposed to 8 X 1 0 ~ 6 M -

Ca with pH buffered at 7-3, 7-5 or 7-6 (with thedissociation constant of the Ca-EGTA complex recal-culated), but no protective effect was noted.

Action of protease inhibitors

The R. pipiens skinned preparations were used for arange of experiments in which various protease inhibi-tors, both singly and in combination, were tested forpossible effects in the reduction of damage. Table 2summarizes the various protocols used and in the first

Figs 9-14. Examples of extreme, Ca-triggered damage insaponin-skinned pectoris cutaneous fibres. Bar, 1 (Jm,except Fig. 14.

Fig. 9. Z-line blurring, contracted fibrils, dissolution ofcellular contents with separation of fibrils, swollen vesicles(probably SR); 10~6M-Ca, 15min; R. temporaries.

Fig. 10. Very marked Z-line blurring, but otherwisesimilar to Fig. 9. 5xlO~5M-Ca, 28 min, R. temporaria.

Fig. 11. Extreme contraction and hypercontractionbands, many swollen vesicles and dividing mitochondria;8xlO"6M-Ca plus 14/igml"1 pepstatin, 25 min; R. pipiens.

Fig. 12. Z-line blurring and hypercontraction withdestruction of myofilament apparatus at high [Ca](5xl0~5M), 28min, R. temporaria.

Fig. 13. Z-line blurring and contracted myofibrils withsections in which the myofilaments are destroyed, leavingcytoskeletal elements; swollen mitochondria; 8XlO~6M-Caplus 14^gml~' pepstatin, 25 min; R. pipiens.

Fig. 14. Central hypercontraction band with progressivedissolution of the myofilaments on one side whilst on theother are markedly swollen vesicles and damagedmitochondria; the myofibrils show early damage in thislatter area but the Z-lines show sliding; 8xlO~6M-Ca,30min; R. temporaria. Bar,

series (experiments 1-6) the preparations were skinnedfor 30 min with saponin at 40^gml~', whereas inexperiments 7 and 8 this procedure was increased to40 min at 45 fig ml"1 so as to increase the severity ofskinning. In both series (experiments 1-8) the inhibi-tor was included in the skinning solution and in the

Figs 15, 16. Ca-triggered changes in the mitochondria insaponin-skinned pectoris cutaneous fibres of/?, temporaria.Bar, 1 ^m.

Fig. 15. Myofibrils largely unaffected and not contractedbut mitochondria swollen, damaged and dividing;8xlO"6M-Ca, 40min.

Fig. 16. Swollen, dividing mitochondria with greatlyswollen vesicles, which may represent SR or, moreprobably in this example, the remnants of mitochondria;contracted myofibrils with blurred Z-lines; 8xlO~6M-Ca,30 min.

Calcium and muscle damage 587

Figs 17-21. Different examples of Ca-triggered damage to the myofilament apparatus in which the Z-lines are blurTed butlargely intact and where they remain held together by longitudinal cytoskeletal elements. Fig. 20 shows intact cells, otherfigures are of saponin-skinned cells.

Fig. 17. Myofibrils remain in contracted position with Z-hnes blurred; 3xlO~7M-Ca, 30min; R. temporaria. Bar,0-5 Jim.

Fig. 18. Myofibrils mostly remain in contracted position with areas of hypercontraction; stretched cytoskeletal elementsshow more clearly than in Fig. 17; 8X 10~6M-Ca, 30min; R. pipiens. Bar, 1 Jim.

Fig. 19. 8X1O"6M-Ca, 30 min; R. pipiens. Bar, 1 Jim.Fig. 20. Intact cell, cytoskeletal elements much stretched by small areas of contracted sarcomeres; lOmM-caffeine,

30min; R. temporaria. Bar, 2/im.Fig. 21. Similar effect to Fig. 20 but in a skinned cell; 8xlO~6M-Ca, 30min; R. temporaria. Bar, 1 jtm.

588 C. jf. Duncan

Table 2. Protease inhibitors tested that did not prevent rapid ultrastructural damage in skinned cutaneouspectoris preparations o/R. pipiens

Exptno.

lalb2a2b2c3

4a4b5

6

7

8

9

Skinning:saponin concn/time

(jig ml"'/min)

40/2540/2540/2540/2540/2540/25

40/2540/2540/25

40/25

45/40

45/40

40/20

Inhibitor(s)

AprotininAprotininLeupeptinLeupeptinLeupeptinChymostatin

PepstatinPepstatinPepstatin + IAA

Pepstatin +leupeptin

Pepstatin +leupeptin

Pepstatin

pCMB/EGTA

Concn(/igmP1)

21330355040

2-514

2 - 5 + 1 0 - 3 M

2-5 + 50

14+30

14

10"3M

Enzymes of degradativepathways inhibited

Proteases

CANP; cathepsins B, H and L

Chymotrypsin; cathepsins B andD; non-lysosomal

Cathepsin D

Cathepsins B, D, L, H andCANP

Cathepsins B, D, L, H + CANP

Cathepains B, D, L, H + CANP

Cathepsin D

Cathepsins B, H, L + CANP

Mean time forvisible damage

(nun)

22256

14

75

15

15

5

7

0-5

10 40/25 None Control 3

After skinning, all preparations (except expt 9) were tested with buffered 8X 10 6M-Ca for 25 min before fixation. Expt 9 has 0 Ca andwas fixed after 5 min. All preparations showed severe ultrastructural damage, including hypercontraction in expts 1-8. Inhibitors wereincluded during skinning, rinsing and exposure to Ca. For details of the action and concentrations of enzyme inhibitors sec Barrett &McDonald (1980), Umezawa (1976). For details of lysosomal and non-lysosomal pathways in hcpatocjtes see Grinde & Seglen (1980). Allexperiments were done in triplicate. IAA, lodoacetic acid.

subsequent rinse in the potassium glutamate mediumand during the test exposure (25 min) to 8X 10~6 M-Ca,a concentration that consistently caused damage incontrol experiments. In experiment 9, the muscleswere skinned in the absence of inhibitor and were thentransferred to l(T3M-pCMB in 0-5mM-EGTA (i.e.zero [Ca]) and the preparation was fixed after only5 min. The inhibitors were chosen to determinewhether CANP or individual lysosomal cathepsinswere implicated in rapid subcellular damage; combi-nations of inhibitors were selected to protect againstpossible lysosomal breakdown and the release of arange of acidic proteases.

In experiments 1-8, transfer to Ca caused rapid,severe and extensive damage, which was usually detect-able by transmitted light within 10 min (Table 2).There were differences between preparations or be-tween experiments in the time before changes wereclearly visible, but no consistent protection was foundin any of the experiments and electron micrographsprepared at the end of the experiment invariablyrevealed hypercontraction bands in addition to theother types of damage (Figs 5, 7, 11, 13). pCMB, an-SH blocker and hence an inhibitor of cysteine pro-teases, itself caused visible damage within 30 s in the

absence of Ca, and electron micrographs of the prep-aration after only 5 min revealed widespread damage,both actin loss and disintegration of myofibrils (cat-egories 1, 4 and 5) and also contraction damage(category 6). Thus, this damaged caused with zero[Ca] appears to be identical with the Ca-triggereddamage.

Action of trifluoperazine (TFP)

One possibility concerning the action of Ca in trigger-ing subcellular damage is that the ubiquitous calmodu-lin may be involved at one step in the degradativesequence and hence the anti-calmodulin drug TFP wastested on the saponin-skinned preparation. Three sep-arate series of experiments with different batches offrogs (R. pipiens and R. temporaria) with appropriateprotocols for skinning (R. temporaria = 60f/gml~'saponin, 30min; R. pipiens = 37 • 5 fug m\~l saponin,30 min) and with pre-exposure to TFP (10~5M or3 X 1 0 ~ 5 M ) for 15 or 20min were used. Concentrationsof TFP were chosen to replicate trials with TFP inintact muscle preparations (Duncan & Rudge, 1984).However, none of these measures provided any protec-tion against the addition of 8xlO~6M-Ca in the pres-ence of TFP; both the time-course of the visual damageand the ultrastructural appearance (damage categories

Calcium and muscle damage 589

1-8) after 30 min exposure were similar to those seen inthe matched controls.

Discussion

Saponin-skinned muscles

The saponin-skinned, cutaneous pectoris preparation(Endo & lino, 1980) proved to be a satisfactory systemfor determining the effect of Ca in initiating cellulardamage, and in this thin sheet of muscle extremedamage can be detected visually by transmitted lightand it has always been subsequently confirmed byelectron microscopy. High concentrations of saponinappeared to be able to promote damage directly,suggesting that the system causing damage is mem-brane-bound and sensitive to membrane disruption.The action of glutaraldehyde-based fixatives in causingvisible damage in skinned preparations almost immedi-ately may also be via a membrane perturbation, as in itsaction on the Ca-ATPase of the SR (Thomas &Hidalgo, 1978).

The R. pipiens and R. temporaria preparationsappeared to be genuinely different in their sensitivity tosaponin and this observation may reflect a difference inthe molecular structure of their muscle membranes.Furthermore, small, but consistent, differences insensitivity between winter and summer frogs were alsofound. These poikilotherms have different optimalenvironmental temperatures and R. pipiens had beenmaintained at 23 °C, whereas R. temporaria was kept at9°C, so that homeoviscous adaptation may well pro-duce differences in the lipid composition of theirmembranes.

The role of Ca

The results show unequivocally that intracellular Cacan trigger rapid damage in saponin-skinned amphib-ian skeletal muscle. The time taken to produce thisdamage is concentration-dependent and in both R.temporaria and R. pipiens the effective concentrationrange is probably about 10~7 to 10~5M-Ca, i.e. ap-proximately the same range as the changes in concen-tration experienced in the cytosol during excitation-contraction-relaxation. As emphasized above, thesaponin-skinned muscle may be partially activated;nevertheless the results do show that rapid damage canbe triggered at very low cytosolic [Ca]. During contrac-tion in the normal muscle cell the release of Ca willproduce local, high levels of [Ca],, but these will betransient and the Ca2+ will be rapidly shuttled betweenthe SR and the troponin C and parvalbumins.

The steady-state level of [Ca], in the muscle cell atrest (approximately 10~7 M) will be the result of passiveinflux and active efflux across the sarcolemma and ofuptake by the SR (the low affinity mitochondrial Ca

uptake is probably not implicated). At normal resting[Ca],, in the closed system of the muscle cell, net activemovement of Ca will be low, [Ca], being close to thelower sensitivity of the pumps for active transport.However, in the skinned muscle cell, immersed in alarge volume of Ca-EGTA buffer, even at low free[Ca], active transport will continue as the pumpsattempt the impossible task of reducing the free [Ca].We conclude that it is not the concentration of Ca thatis important in triggering myofilament damage, butrather the duration and magnitude of active movementof Ca across key membranes. In muscles where thesteady-state level of [Ca], is elevated, perhaps quitemodestly (e.g. in dystrophic muscle), damage may betriggered by the continued active transport of Ca over aperiod of many minutes.

Damage can be detected visually after 90 s in skinnedpreparations at 8xlO~6M-Ca and this rapidity is con-sistent with results obtained with rat hearts in Ca-paradox experiments. Return of extracellular Ca afterCa-free perfusion caused the typical loss of cellularproteins but, significantly, if the hearts were repletedwith Ca for only 30 s and then returned to Ca-freeperfusion, the resultant protein loss was the same(Hunt & Willis, 1985).

The role of Sr

The action of Sr-EGTA in triggering damage, albeit athigher concentrations (effective range 3xlO~6M to5 X 1 0 ~ 4 M ) , is in accord with observations on intactpreparations, particularly amphibian and mammalianhearts where Sr can substitute for Ca in triggeringdamage (DeLeiris & Feuvray, 1973; Goshima et al.1978) that was ultrastructurally identical (Rudge,1983).

Ultrastructural damage in intact and skinnedcutaneous pectoris

The damage produced in the skinned cells is summar-ized in Table 1 and can be classified into two differenttypes of response. First, loss of actin filaments, break-ing and disintegration of the myofibrils, and myofila-ments separating from the Z-line (categories 1, 3 and4); this type of damage is characterized by sharp andclearly defined Z-lines. Second, categories 6 and 7appear to accompany contraction of the myofilamentapparatus and concomitantly the Z-line widens andbecomes blurred (see Figs 10, 11, 12, 17), particularlyin areas of hypercontraction. In areas of hypercontrac-tion, the myofilament apparatus appears to be pro-tected from disassembly and thus a sarcomere mayrespond with one of the two types of damage. Thepatterns of damage found in skinned cells closelyresemble those found in intact cutaneous pectoristreated with A23187 (Statham et al. 1976). Hypercon-traction bands and dramatic mitochondrial subdivision

590 C. J. Duncan

are also characteristically found in damaged amphibianand mammalian cardiac muscle and in mammalianskeletal muscle in which [Ca]; has been raised (Publi-cover et al. 1978; Duncan & Smith, 1980; Duncan etal. 1980; Rudge & Duncan, 1984a,fc).

Thus, we conclude (1) that the characteristic andcommon patterns of rapid damage (both in vivo and invitro) in vertebrate skeletal and cardiac muscle reflect acommon underlying sequence of cellular events thatculminate in specific ultrastructural degradation of themyofilaments; (2) that the damage caused by A23187 inintact amphibian (StathameJa/. 1976) and mammalian(Publicover et al. 1978) skeletal and cardiac (Rudge &Duncan, 1980, 1984a) muscles are indeed triggered byrises in [Ca], and in Ca fluxes in the cell.

Ruthenium Red and the regulation of [Ca]j

Ruthenium Red causes rapid and characteristic damagein intact cardiac and skeletal muscles (Duncan et al.1980; Rudge & Duncan, 1980, 1984a), but was ineffec-tive in the skinned preparation. We conclude that in theintact cell Ruthenium Red inhibits the Ca-ATPases ofthe Ca pumps of the sarcolemma, SR or mitochondriaand thereby raises [Ca], and so, in turn, initiatesmyofilament damage.

Ultrastructural damage in the absence of CaThe experiments with pCMB in the presence ofEGTA, which cause the same characteristic myofila-ment damage (presumably via an action on key -SHgroups), demonstrate that an alteration in [Ca]; is notthe only means whereby the underlying cellular mech-anism can be initiated.

Role of calmodulin

Previous in vitro experiments with trifluoperazine(TFP) and intact skeletal and cardiac muscles showedthat not only did this anti-calmodulin agent fail toprotect against the characteristic ultrastructuraldamage produced by a variety of different means, butthat exposure to 10~5 M-TFP alone produced identicalpatterns of damage (Duncan & Rudge, 1984). Clearly,calmodulin could act to modify either the systemsregulating [Ca], or the Ca-triggered damage processitself. Since TFP does not protect against Ca-induceddamage in the skinned cell in the present experiments,we conclude that calmodulin does not have an overrid-ing control of the rapid damage process that proceedsin the presence of TFP. Presumably, TFP in vitroinhibits the calmodulin-activation of the Ca-ATPase ofthe sarcolemma thereby raising [Ca](.

Mechanisms of cellular damage

Previously, muscle damage has been measured eitherqualitatively by histological or ultrastructural assess-ment, or quantitatively by assaying the release ofcytoplasmic proteins, a method that reflects only the

lack of integrity of the sarcolemma. We do not yet knowthe sequence of events in cellular damage, but recentstudies (Duncan & Jackson, 1987) have shown that atleast two independent pathways are involved: (1) PLA2(phospholipase A2) activation and subsequent lipoxy-genase activity culminating in sarcolemma damage;and (2) a separate system that produces the character-istic destruction of the myofilament apparatus.

A number of theories are extant concerning themechanisms underlying cellular damage but the pres-ent studies with skinned fibres show that some of theseare not implicated in the specific and rapid damage ofthe myofilaments and these are discussed below.

Intracellular pH. pH, falls markedly in malignanthyperthermia and in cardiac ischaemia, with irre-versible cell injury occurring at pH6-3 (Gebert et al.1971; Ichiharae/ al. 1979; Schaper & Knoll, 1979). Itis proposed that protons are emitted in exchange for Cauptake by the SR and mitochondria, that acidificationalso results from ATP breakdown (Grinwald & Nayler,1981) and that the resulting acidosis injures cells andcauses membrane damage (Ganote, 1983). However,'intracellular' pH is firmly buffered in the skinned cellsand no protective effect was found even when the pHwas raised to 7-3, 7-5 or 7-6, supporting the results ofstudies with intact muscle cells showing that rapiddamage is triggered even when the mitochondria areuncoupled (Duncan et al. 1980).

ATP deletion. During rapid damage, in both skeletal(in malignant hyperthermia) and cardiac (in ischaemia;Ganote, 1983) muscles, the levels of high-energyphosphates fall dramatically. It is proposed that thisprocess directly causes damage and rigor complexesand also permits alterations in membrane permeability(Jennings & Reimer, 1981) or changes in electrolytelevels (Nayler, 1981) to occur.

However, rapid damage in less than 120 s wastriggered by Ca in the skinned muscle with 3 mM-ATPand it is unlikely that high-energy phosphate depletionis the direct cause of myofilament damage.

Calcium-activated neutral protease. Since CANPwas localized in skeletal and cardiac muscles, consider-able work has been directed towards elucidating itspossible role in myofibrillar degradation. CANP is alsoactivated by Sr and its properties and role in degra-dation in muscles have been summarized by Imahori(1982) and Ishiura et al. (1982).

However, it is inhibited by leupeptin, iodoacetate,pCMB and EGTA, and none of these agents preventedrapid Ca-activated damage or pCMB-induced damagein skinned muscle preparations (Table 2), confirmingprevious conclusions from studies with intact skeletal(Duncan et al. 1979) and cardiac (Rudge & Duncan,1984a) muscle that CANP is not the major element inrapid cellular damage.

Calcium and muscle damage 591

CANP and the cytoskeletal lattice. Titin and nebu-lin (or connectin) form an elastic network withinmuscle cells and both are rapidly cleaved by endogen-ous and exogenously added proteases, especially CANP(Wang, 1982). If the cytoskeletal remnant9 left afterrapid cellular damage (Figs 17-21) are indeed a resist-ant titin-nebulin network, these observations provideconfirmatory evidence that CANP is not involved. It isthus also unlikely that lysosomal proteases (see below)are responsible for rapid myofilament degradation andit seems that this damage, stimulated by pCMB, maybe a preferential attack on -SH-linked subunits, sincetitin and nebulin are believed to be constructed from(non-disulphide) covalently linked subunits (Wang,1982).

Lysosomal breakdown. Since lysosomal cathepsinsB and D degrade myofibrillar proteins (Schwartz &Bird, 1977; Matsumoto et al. 1983), a number ofworkers have suggested that cellular degradation isproduced by the release of these enzymes (Kennett &Weglicki, 1978; Wildenthal & Crie, 1980; Katanuma &Noda, 1982), and the fall in pH; (see above) wouldpermit their activity once free in the cytosol. Lyso-somal enzyme activity has been reported to be elevatedin various muscular dystrophies (Pearson & Kar, 1979;Noda et al. 1981) and in ischaemic heart (Wildenthal,1978).

Studies with protease inhibitors on intact muscle arebedevilled by uncertainty as to whether they canpenetrate the sarcolemma, but none of the inhibitorstested on skinned muscles in this study prevented rapidmyofilament damage, nor were any differences fromnormal, characteristic damage noted, even when theskinning procedure was extended (Expts 7 and 8, Table2). Cathepsin D is inhibited by pepstatin and chymo-statins; cathepsins B, H and L are inhibited by thiol-blocking agents, iodoacetate and leupeptin. The mix-tures of inhibitors (Expts 5-7, Table 2) were chosen toprotect against the release of mixed lysosomal pro-teases. It is also unlikely that acidic proteases couldachieve complete degradation in 90 s at pH 7-5, and it isevident that lyososmal cathepsins are not involved inrapid myofilament degradation in amphibian skeletalmuscle.

To conclude, low levels of Ca trigger rapid andcharacteristic myofilament damage in skinned skeletalmuscle cells. Neither a fall in pH;, a fall in high-energyphosphate reserves, activation of CANP nor the releaseof lysosomal enzymes is directly involved, although allmay have a peripheral role in vivo.

The assistance of Mr J. L. Smith with the electronmicroscopy and of Miss S. Scott in preparing the manuscriptis gratefully acknowledged.

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