Different mechanisms mediate structural changes and intracellular enzyme efflux following damage to skeletal muscle

0 Department of Medicine, University of Liverpool , PO Box 147, Liverpool L69 3BX , UK - Author for correspondence Cellular damage, induced by either A23187 (2xl(TsM) or 2,4-dinitrophenol (DNP) (1(T3M), 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~4M; an inhibitor of phospholipase A (PLA2)) or 5xlO~6M-nordihydroguaiaretic 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) PLA2 activation and lipoxygenase activity culminating in sarcolemma damage and (2) a system that produces characteristic destruction of the myofllament apparatus. 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 transplant 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 scavengers (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 dystrophy (Duncan, 1978; Rodemann et al. 1981; Jones et al. 1983). Unfortunately, the majority of such studies have examined only one indicator of damage. The most popular of these have been protein degradation 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 nondiffusible 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 (PLA2) 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 determine 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. Materials and methods Female Balb/C mice maintained on a standard laboratory diet were killed by cervical dislocation and the soleus muscles were rapidly dissected and removed. The muscles were 10 mm long and the tendons were ligatured, then attached to glass holders and placed in separate plastic tubes containing 2-5 ml of bicarbonate-buffered Krebs saline. The tubes were mounted in a thermostatically controlled water bath at 37C (Jones et al. 1983). The incubation medium contained 137mM-NaCl, 5-0mM-KCl, l-0mM-MgSO4, l-3mM-NaH2PO4, 24-0mM-NaHCO3l 2-0 mM-CaCl2, 10-OmM-glucose and was gassed with 95% O2, 5% CO2 (pH7-4). After 30min incubation the medium surrounding the muscles was exchanged and agents known to damage muscles and induce enzyme efflux were added to the medium. After 30 min the medium was exchanged and this was repeated every 30 min until the end of the experiment. The creatine kinase (CK) activity of the incubation medium was measured by using a linked enzyme assay, the production of NADPH being followed at 340 nm (Jones et al. 1983). When inhibitors of enzyme efflux were used they were added to the incubation medium and the preparations were preincubated for 30 min before the addition of A23187 or 2,4-dinitrophenol (DNP). In media lacking Ca, CaCl2 was 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 tension for fixation in 3 % glutaraldehyde at 21 C (15 h) for electron microscopy. Details of subsequent washing, dehydration and sectioning have been given by Duncan et al. (1980) and sections at 6090 nm were examined on a Zeiss EM10/CR. All agents used were of AnalaR grade or the highest grade commercially available. Calcium ionophore (A23187), DNP, chlorpromazine and nordihydroguaiaretic acid (NDGA) were obtained from Sigma Chemical Company, Poole, Dorset. A23187 was solubilized in absolute ethanol and an equivalent volume of ethanol was included in control preparations. Effects of calcium ionophore on muscle CK efflux The CK efflux from a group of muscles treated with the calcium ionophore A23187 (20 ^M) are shown in Fig. 1, together with the CK efflux from muscles treated with calcium ionophore in the presence of chlorpromazine (200/iM) or NDGA (5fiM). Treatment of muscles with ionophore induced a large and rapid release of this intracellular enzyme, which was almost completely prevented by the presence of chlorpromazine or NDGA. Effect of DNP on muscle CK efflux DNP ( 1 0 ~ 3 M ) treatment induced a large and rapid efflux of CK from muscles (Fig. 2), which was almost completely eliminated by removal of calcium from the medium bathing the muscles, showing that the C. J. Duncan and M. jf. Jackson Fig. 1. Efflux of creatine kinase from mouse soleus muscles incubated in the presence of calcium ionophore (A23187). Muscles were incubated for 30min and the incubation medium was then exchanged (time 0) and calcium ionophore (20^M) added for 30 min. The muscles were then incubated for a further 30 min in incubation medium 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 with chlorpromazine or NDGA ( ) . increased permeability of the sarcolemma induced by D N P in the soleus preparation is dependent on Ca influx. Similarly, the inhibitors chlorpromazine (200 fM) or N D G A (50 (JM), in the presence of extracellular Ca, provided almost complete protection against C K efflux. Ultrastructural studies of muscle damage Control muscles fixed either at the start of the experiment or after incubation for 60 min revealed a normal ultrastructure (Fig. 3), confirming that the conditions of incubation were satisfactory. Exposure of soleus muscle to (...truncated)