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Different mechanisms mediate structural changes and intracellular enzyme efflux following damage to skeletal muscle C. J. DUNCAN 1 '* and M. J. JACKSON 2 1 Department of Zoology and 1 Department of Medicine, University of Liverpool, PO Box 147, Liverpool L69 3BX, UK •Author for correspondence Summary Cellular damage, induced by either A23187 (2xl(T s M) or 2,4-dinitrophenol (DNP) (1(T 3 M), has been studied in incubated mouse soleus muscle and has been monitored by electron microscopy and measurement of creatine kinase (CK) efflux. CK efflux induced by DNP was dependent on extracellular Ca, whereas the characteristic myofllament breakdown was not. Chlorpromazine (2xlO~ 4 M; an inhibitor of phospholipase A (PLA 2 )) or 5xlO~ 6 M-nordi- hydroguaiaretic acid (NDGA) (a lipoxygenase inhibitor) almost completely inhibited CK efflux, following treatment with either A23187 or DNP, but did not affect myofilament damage. It is concluded that there are at least two separate pathways in cellular damage: (1) PLA 2 activation and lipoxygenase activity culminating in sarco- lemma damage and (2) a system that produces characteristic destruction of the myofllament apparatus. Key words: muscle damage, phospholipase A2, lipoxygenase, calcium. Introduction Damage to tissues is known to occur in a variety of pathological conditions and a considerable amount of work has been undertaken in order to elucidate the mechanisms by which different stresses induce such damage. It has been assumed that a knowledge of the various processes involved in tissue damage would allow possible therapeutic intervention and hence the maintenance of tissue viability. In particular, the processes involved in damage to cardiac muscle have been extensively studied, with the result that the viability of cardiac tissue either removed for trans- plant purposes or subjected to major surgery in vivo can be improved by using agents such as calcium antagonists (Nayler et al. 1979) or free radical scav- engers (McCord, 1985). Skeletal muscle has also been studied with a view to finding agents capable of reducing muscle damage in chronic degenerative disorders such as muscular dys- trophy (Duncan, 1978; Rodemann et al. 1981; Jones et al. 1983). Unfortunately, the majority of such studies have examined only one indicator of damage. Journal of Cell Science 87, 183-188 (1987) Printed in Great Britain © The Company of Biologists Limited 1987 The most popular of these have been protein degra- dation rates (Rodemann et al. 1981; Baracos et al. 1986), ultrastructural appearance (Publicover et al. 1978) and efflux of intracellular enzymes (Jones et al. 1983; Jackson et al. 1984) or exclusion of non- diffusible compounds, e.g. Trypan Blue (Zuurveld et al. 1985). A number of compounds have been described that reduce enzyme efflux from skeletal muscle and from which conclusions concerning the mechanism of release of intracellular enzymes have been drawn (Jackson et al. 1983, 1984). In particular, inhibitors of phospholipase A2 (PLA 2 ) and of the enzymic oxidation of arachidonic acid via the lipoxygenase pathway (Jackson et al. 1987) have proved to be effective, leading to the suggestion that these pathways are implicated in sarcolemma breakdown. The aim of this work was to use chlorpromazine (an inhibitor of phospholipase enzymes) and nordihydroguaiaretic acid, NDGA (an inhibitor of lipoxygenase), to de- termine whether the characteristic ultrastructural changes that are seen during skeletal muscle damage occur via the same mechanism as the efflux of intracellular enzymes following a damaging stress. 183

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Page 1: Different mechanisms mediate structural changes and ... · Different mechanisms mediate structural changes and intracellular enzyme efflux following damag toe skeletal muscle C. J

Different mechanisms mediate structural changes and intracellular

enzyme efflux following damage to skeletal muscle

C. J. DUNCAN1'* and M. J. JACKSON2

1Department of Zoology and 1Department of Medicine, University of Liverpool, PO Box 147, Liverpool L69 3BX, UK

•Author for correspondence

Summary

Cellular damage, induced by either A23187(2xl(TsM) or 2,4-dinitrophenol (DNP) (1(T3M),has been studied in incubated mouse soleusmuscle and has been monitored by electronmicroscopy and measurement of creatine kinase(CK) efflux. CK efflux induced by DNP wasdependent on extracellular Ca, whereas thecharacteristic myofllament breakdown was not.Chlorpromazine (2xlO~4M; an inhibitor ofphospholipase A (PLA2)) or 5xlO~6M-nordi-hydroguaiaretic acid (NDGA) (a lipoxygenase

inhibitor) almost completely inhibited CK efflux,following treatment with either A23187 or DNP,but did not affect myofilament damage. It isconcluded that there are at least two separatepathways in cellular damage: (1) PLA2 activationand lipoxygenase activity culminating in sarco-lemma damage and (2) a system that producescharacteristic destruction of the myofllamentapparatus.

Key words: muscle damage, phospholipase A2,lipoxygenase, calcium.

Introduction

Damage to tissues is known to occur in a variety ofpathological conditions and a considerable amount ofwork has been undertaken in order to elucidate themechanisms by which different stresses induce suchdamage. It has been assumed that a knowledge of thevarious processes involved in tissue damage wouldallow possible therapeutic intervention and hence themaintenance of tissue viability. In particular, theprocesses involved in damage to cardiac muscle havebeen extensively studied, with the result that theviability of cardiac tissue either removed for trans-plant purposes or subjected to major surgery in vivocan be improved by using agents such as calciumantagonists (Nayler et al. 1979) or free radical scav-engers (McCord, 1985).

Skeletal muscle has also been studied with a view tofinding agents capable of reducing muscle damage inchronic degenerative disorders such as muscular dys-trophy (Duncan, 1978; Rodemann et al. 1981; Joneset al. 1983). Unfortunately, the majority of suchstudies have examined only one indicator of damage.

Journal of Cell Science 87, 183-188 (1987)Printed in Great Britain © The Company of Biologists Limited 1987

The most popular of these have been protein degra-dation rates (Rodemann et al. 1981; Baracos et al.1986), ultrastructural appearance (Publicover et al.1978) and efflux of intracellular enzymes (Joneset al. 1983; Jackson et al. 1984) or exclusion of non-diffusible compounds, e.g. Trypan Blue (Zuurveld etal. 1985).

A number of compounds have been described thatreduce enzyme efflux from skeletal muscle and fromwhich conclusions concerning the mechanism ofrelease of intracellular enzymes have been drawn(Jackson et al. 1983, 1984). In particular, inhibitorsof phospholipase A2 (PLA2) and of the enzymicoxidation of arachidonic acid via the lipoxygenasepathway (Jackson et al. 1987) have proved to beeffective, leading to the suggestion that these pathwaysare implicated in sarcolemma breakdown. The aim ofthis work was to use chlorpromazine (an inhibitorof phospholipase enzymes) and nordihydroguaiareticacid, NDGA (an inhibitor of lipoxygenase), to de-termine whether the characteristic ultrastructuralchanges that are seen during skeletal muscle damageoccur via the same mechanism as the efflux ofintracellular enzymes following a damaging stress.

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Materials and methods

Female Balb/C mice maintained on a standard laboratorydiet were killed by cervical dislocation and the soleusmuscles were rapidly dissected and removed. The muscleswere 10 mm long and the tendons were ligatured, thenattached to glass holders and placed in separate plastictubes containing 2-5 ml of bicarbonate-buffered Krebssaline. The tubes were mounted in a thermostaticallycontrolled water bath at 37°C (Jones et al. 1983). Theincubation medium contained 137mM-NaCl, 5-0mM-KCl,l-0mM-MgSO4, l-3mM-NaH2PO4, 24-0mM-NaHCO3l 2-0mM-CaCl2, 10-OmM-glucose and was gassed with 95% O2,5% CO2 (pH7-4). After 30min incubation the mediumsurrounding the muscles was exchanged and agents knownto damage muscles and induce enzyme efflux were added tothe medium. After 30 min the medium was exchanged andthis was repeated every 30 min until the end of the exper-iment. The creatine kinase (CK) activity of the incubationmedium was measured by using a linked enzyme assay, theproduction of NADPH being followed at 340 nm (Jones etal. 1983). When inhibitors of enzyme efflux were used theywere added to the incubation medium and the preparationswere preincubated for 30 min before the addition of A23187or 2,4-dinitrophenol (DNP). In media lacking Ca, CaCl2was simply omitted and EGTA was not included so that[Ca]o was 3X106 to 6x 1 0 " 6 M .

Muscles were mounted on a frame under very light tensionfor fixation in 3 % glutaraldehyde at 21 °C (15 h) for electronmicroscopy. Details of subsequent washing, dehydrationand sectioning have been given by Duncan et al. (1980) andsections at 60— 90 nm were examined on a Zeiss EM10/CR.

All agents used were of AnalaR grade or the highestgrade commercially available. Calcium ionophore (A23187),DNP, chlorpromazine and nordihydroguaiaretic acid(NDGA) were obtained from Sigma Chemical Company,Poole, Dorset. A23187 was solubilized in absolute ethanoland an equivalent volume of ethanol was included in controlpreparations.

Results

Effects of calcium ionophore on muscle CK effluxThe CK efflux from a group of muscles treated withthe calcium ionophore A23187 (20 ^M) are shown inFig. 1, together with the CK efflux from musclestreated with calcium ionophore in the presence ofchlorpromazine (200/iM) or NDGA (5fiM). Treat-ment of muscles with ionophore induced a large andrapid release of this intracellular enzyme, which wasalmost completely prevented by the presence of chlor-promazine or NDGA.

Effect of DNP on muscle CK efflux

DNP ( 1 0 ~ 3 M ) treatment induced a large and rapidefflux of CK from muscles (Fig. 2), which was almostcompletely eliminated by removal of calcium fromthe medium bathing the muscles, showing that the

150

3E

I 100

50

o

0 30 60Time after addition of A23187 (min)

Fig. 1. Efflux of creatine kinase from mouse soleusmuscles incubated in the presence of calcium ionophore(A23187). Muscles were incubated for 30min and theincubation medium was then exchanged (time 0) andcalcium ionophore (20^M) added for 30 min. The muscleswere then incubated for a further 30 min in incubationmedium alone. Results are presented as mean ± S.E.M.(n = 4) for muscles treated with ionophore alone ( • ) ,and as mean of two muscles for those treated withchlorpromazine « » or NDGA ( • ) .

increased permeability of the sarcolemma inducedby D N P in the soleus preparation is dependent onCa influx. Similarly, the inhibitors chlorpromazine(200 fM) or N D G A (50 (JM), in the presence ofextracellular Ca, provided almost complete protectionagainst CK efflux.

Ultrastructural studies of muscle damage

Control muscles fixed either at the start of the exper-iment or after incubation for 60 min revealed a normalultrastructure (Fig. 3), confirming that the conditionsof incubation were satisfactory. Exposure of soleusmuscle to either A23187 or DNP produced the samecharacteristic pattern of myofilament damage (Figs 4,5, 7) found in diaphragm muscle exposed to theseagents (Publicover et al. 1978; Duncan et al. 1980);namely, (1) Z-line sliding (Figs 4, 5), Z-line blurringor complete Z-line loss (Figs 7, 8); (2) focal myofila-ment apparatus loss (Fig. 4); (3) disruption of themyofilament apparatus, separating from the Z-line,culminating in disintegration and dissolution of thethick and thin filaments (Figs 5, 6); (4) contraction

184 C. J. Duncan and M. jf. Jackson

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18.1 200

3

V

C'Si

100

u

zD

0 30 60Time after addition of DNP (min)

Fig. 2. Effect of removal of extracellular calcium (O), oraddition of chlorpromazine (O) o r NDGA (B) on theefflux of creatine kinase from muscles treated with DNP,compared with muscles treated with DNP alone (9).Results are presented as the mean ± S.E.M. of four musclestreated with DNP alone and the mean of two muscles forthose treated to reduce enzyme efflux.

(Fig. 5) (particularly associated with Z-line blurring);(5) hypercontraction bands; (6) mitochondrialchanges including swelling, division and the develop-ment of 'bars' within the organelle (see Fig. 8). Thedifference between the damage triggered by eitherA23187 or DNP in mouse diaphragm and soleus wasthat in the latter preparation the damage after 30 minexposure was largely confined to the outer fibres.

However, in all of the protocols summarized inFigs 1 and 2, where the inhibitors provided almostcomplete protection against CK efflux, identical pat-terns of myofilament damage were found at the end ofthe experiments. In summary, neither NDGA norchlorpromazine protected against A23187 (Fig. 8) orDNP-induced ultrastructural damage (Fig. 6). All thedifferent features of myofilament damage and otherultrastructural changes listed above, including swell-ing of the sarcoplasmic reticulum and mitochondrialdivision, were found in experiments when chlorpro-mazine or NDGA was included in the incubationmedium.

Furthermore, whereas omission of extracellular Caprotected against DNP-induced CK efflux (Fig. 2),

the same concentration of DNP caused severe myofila-ment damage in both the presence and absence ofextracellular Ca (see Figs 4, 5).

Discussion

Evaluation of studies examining the mechanisms ofdamage to skeletal muscle is hampered by a lack ofinformation on the relationship between the variousindices of damage used by different workers. Forexample, treatment of skeletal muscle with the cal-cium ionophore (A23187) has been shown to raiseintracellular calcium levels, inducing ultrastructuraldamage (Publicover et al. 1978), intracellular enzymeefflux (Jones et al. 1984), an increase in proteindegradation rates (Rodemann et al. 1981) and a loss ofTrypan Blue exclusion (Zuurveld et al. 1985), butpretreatment of muscles with the protease inhibitorleupeptin has been reported to reduce the calcium-stimulated increase in protein degradation (Rodemannet al. 1981), but not calcium-induced ultrastructuraldamage (Duncan et al. 1979) or intracellular enzymeefflux (Jackson et al. 1984). The work described herehas examined the relationship between ultrastructuraldamage and enzyme efflux from skeletal muscle, anddemonstrates that certain agents known to inhibit theefflux of intracellular enzymes had little effect on theultrastructural changes in the same muscle.

Both the calcium ionophore and DNP produced asimilar characteristic pattern of rapid myofilamentdamage in soleus muscle (Figs 3-8) to that found inmouse diaphragm (Publicover et al. 1978; Duncan etal. 1980), with the major difference that in the soleusmuscle the damage was largely confined to the outerfibres.

It has been suggested that in many situations aninflux of external calcium is an essential part of theprocess that leads to efflux of intracellular enzymes(Jackson et al. 1984; Jones et al. 1984; Claremont etal. 1984) and calcium-induced ultrastructural damageto muscle has been extensively described (Publicoveret al. 1978; Duncan et al. 1980). In 1984 Jones et al.demonstrated that both the intracellular enzyme effluxand ultrastructural damage induced by excessive con-tractile activity could be inhibited by removal ofcalcium from the external medium. The results pre-sented here confirm that external calcium is essentialfor the enzyme efflux that follows DNP treatment(Fig. 2), but they also show that removal of Ca has noeffect on the DNP-induced ultrastructural damage.This observation suggests that ultrastructural damageinduced by DNP is triggered either solely by intra-cellular calcium released from the mitochondria or(more probably, Duncan & Rudge, unpublished) bythe direct activation of the myofilament damage pro-cess by DNP.

Mechanisms of muscle damage 185

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* . • ' . ' • • * » • ' • •' • * : • • ? '

Figs 3-6. Electron micrographs of mouse soleus muscle fixed after 60 min exposure to the various treatments summarizedin the legends to Figs 1 and 2. Bars, 1/irn. Fig. 3, control muscle. Figs 4-6, exposure to 10~3M-DNP; Fig. 4, with zeroextracellular [Ca], notice focal myofilament damage with Z-line sliding; Fig. 5, plus normal extracellular [Ca], notice areaof contraction grading into myofilament destruction and Z-line destruction grading into an area of dissolution ofmyofilaments; Fig. 6, plus normal extracellular [Ca] plus 50/iM-NDGA, notice sarcomeres with Z-line loss and swollenmitochondria grading into an area of complete cellular dissolution.

186 C. jf. Duncan and M. J. Jackson

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Fig8 7, 8. Exposure of mouse soleus to 20/IM-A23187; bars, 1/im. Fig. 7, A23187 alone, notice loss of Z-line and actinfilaments and swollen mitochondria with the early development of 'bars'; Fig. 8, A23187 plus S^M-NDGA, noticesimilarity to Fig. 7 with further development of mitochondnal bars and with myosin dissolution beginning in addition toactin and Z-line loss.

The results presented here confirm the fact thatboth NDGA and chlorpromazine have a substantialprotective effect against intracellular enzyme efflux,but are ineffective against the ultrastructural changes.Chlorpromazine is an agent that has many properties,but which is an extremely efficient inhibitor of PLA2,while NDGA is a potent antioxidant and lipoxygenaseinhibitor, and their lack of effect against the specificultrastructural damage to the myofilament apparatussuggests that activation of PLA2 and the oxidation ofthe released arachidonic acid are not involved in theprocess of ultrastructural damage in a similar mannerto that proposed for the mechanism of enzyme efflux(Jackson et al. 1984, 1985, 1987). We conclude thatthere are likely to be at least two separate mechanismsthat can operate during rapid subcellular damage inmammalian skeletal muscle. First, a rise in [Ca];activates PLA2 and the consequent oxidation ofarachidonic acid culminates in sarcolemma damageand the release of CK. Second, a process can beswitched on by rises in [Ca],, which initially can causefocal myofilament damage in the centre of the cell(Fig. 4). However, results to be presented separatelysuggest that this system is not obligatorily dependenton Ca, but can also be triggered directly by otheragents (Duncan, unpublished data).

This finding of at least two different mechanismsthat mediate enzyme efflux and ultrastructural damagefollowing treatment with a damaging agent receivessupport from clinical observations during therapeuticintervention in chronic myopathies. In Duchennemuscular dystrophy it has been reported that treat-ment with corticosteroids reduces the plasma creatinekinase activity of the patients without significant effecton the course of the disease (Drachman et al. 1974),while in patients with polymyositis treated with ster-oids there may be a significant discrepancy betweenthe plasma creatine kinase activity and the clinicalcourse of the disease (Edwards et al. 1979). Steroidsare known to act to inhibit phospholipase enzymes(Blackwell & Flower, 1983) and so may be analogousin their action to the substances (e.g. chlorpromazine)used in this study.

Despite our findings of apparently disparatemechanisms underlying the two processes of muscledamage, both were activated by either A23187 or DNPand it is probable that the two mechanisms are linkedin various ways. In particular, it is likely that changesin sarcdlemmal permeability that are sufficient to allowthe leakage of large intracellular enzymes will lead alsoto ultrastructural damage to the muscle via an associ-ated influx of extracellular calcium. However, the data

Mechanisms of muscle damage 187

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presented here indicate that the reverse situation, i.e.ultrastructural damage without intracellular enzymeefflux, can also exist in skeletal muscle in vitro.

We thank Miss L. Burns and Mr J. L. Smith for theirexcellent technical assistance, Professor R. H. T. Edwardsfor helpful discussions, Miss S. Scott for assistance in thepreparation of the manuscript, and the Muscular DystrophyGroup of Great Britain and F. Hoffman La-Roche and Co.for financial support.

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(Received 2 October 1986 -Accepted 5 November 1986)

188 C. J. Duncan and M. J. Jackson