Role of calcium in triggering rapid ultrastructural damage in muscle: a study with chemically skinned fibres

There is considerable current interest in the triggering and regulation of cellular breakdown and death in a variety of tissues including brain, liver, muscle and kidney as well as in insects and amphibians undergoing metamorphosis. A range of different factors have been implicated, e.g. lysosomal and non-lysosomal proteases, energy-dependent and energy-independent proteases, cellular supply of high-energy phosphates, thyroid hormones, impaired mitochondrial functioning, prostaglandins, phospholipase A2 activity, endotoxaemia, vitamin E deficiency, oxygen radicals and trauma.

The suggestion that an alteration in intracellular Ca homeostasis was an important step in the cascade of events that culminate in cellular damage (Duncan, 1978; Schanne et al. 1979; Katz & Reuter, 1979; Farber, 1981; Trump et al. 1981; Ishiura, 1981; Nayler, 1983) provided a unifying hypothesis with important medical implications for many degradative conditions. However, in opposition to this hypothesis, the following conclusions have recently been advanced: “chemical-induced hepatic cell death is not caused by an increase in total cellular Ca²⁺ resulting from an influx of extracellular Ca²⁺” (Fariss & Reed, 1985), and “We report here that free Ca in metabolically-poisoned myocytes is remarkably stable and that severe injury to the cell occurs before the free Ca concentration rises above 1 to 3×10⁻⁷M, hence cell damage seems to be a cause, not a consequence of a rise in free Ca” (Cobbold & Bourne, 1984).

It is well known that subcellular damage in both skeletal and cardiac muscle can be produced rapidly, i.e. with a time-course of minutes; e.g. the ischaemic mammalian heart and skeletal muscle during malignant hyperthermia. These clinical events can be replicated experimentally not only in vivo, as in studies of the calcium paradox and ischaemia of cardiac muscle, but also in vitro, as in ultrastructural studies of both skeletal and cardiac muscle. Many of these examples of rapid subcellular damage are triggered by agents that appear to raise [Ca] ₁, in the muscle (both cardiac and skeletal) or to promote Ca fluxes: (1) the Ca paradox of cardiac muscle (Grinwald & Nayler, 1981; Opie, 1985); (2) the actions of the divalent cation ionophore A23187 (Statham et al. 1976; Publicover et al. 1978) where ultrastructural damage has now been linked to rises in [Ca] ₁ (Yoshimura et al. 1986); (3) the action of Dantrolene sodium in preventing the release of Ca from the sarcoplasmic reticulum (SR) and protecting against malignant hyperthermia (Blanck & Gruener, 1983); (4) the effect of the withdrawal of extracellular Ca in protecting against protein efflux from isolated mouse muscle when challenged with excessive contractile activity in anoxia (Jones et al. 1984).

The conclusion from these studies is that abrupt rises in intracellular Ca are able to trigger rapid and characteristic ultrastructural damage and that there is a common degradative mechanism of cardiac and skeletal muscle in both amphibians and mammals.

However, we have, as yet, no direct measurements of intracellular [Ca] during the process of damage in skeletal muscle and thus the first objective of the present study was to develop a chemically skinned skeletal muscle fibre preparation in which intracellular Ca could be accurately controlled. It is shown that a [Ca] of 10⁻⁶M can consistently trigger rapid and characteristic damage.

Second, the skinned fibre preparation was used to assess the importance of proteolytic enzymes in cellular damage. One hypothesis concerns the involvement of Ca-activated neutral proteases (CANP) and, alternatively, the Ca-activated damage process might involve lysosomal labilization and breakdown. Thus, DNP is one agent that is also known to cause release of lysosomal hydrolases only via mechanisms that operate in the intact cell (Martini, 1959), whilst an increase in lysosomal catheptic enzyme activity has been reported in various muscular dystrophies (Pearson & Kar, 1979). It is reported here that a variety of protease inhibitors failed to prevent rapid damage in the skinned muscle cell. Finally, other mechanisms have been suggested concerning the triggering of damage in muscle cells, such as a fall in pH_i or a severe depletion of high-energy phosphates. The skinned-fibre preparation was used to adjust and control the intracellular environment and to test these hypotheses.

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

The preparations were transferred to one of the two following media of composition (mM): (1) sucrose (250), piperazine-N,N’-bis[2-ethanesulphonic acid] (4), magnesium acetate (4), EGTA (0·5), ATP (Na salt) (3), brought to pH 7·1 with NaOH and KOH so that [K⁺] = 2·5 and [Na⁺] = 10. (2) Potassium glutamate (100), piperazine-N,N’-bis[2-eth-anesulphonic acid] (10), magnesium acetate (4), EGTA (0·5), ATP (Na salt) (3), pH7·1 with KOH. Temperature, 18°C. Saponin was added to the skinning solution immediately before use and briskly stirred.

Exposure of skinned preparations to test solutions: skinned preparations were rinsed in ice-cold potassium glutamate solution (medium (2)) and then transferred to fresh potassium glutamate medium (18°C). Samples of 1 M-CaCl ₂ (AnalaR) or of SrCb were added to produce calculated fixed free divalent cation concentrations in the EGTA buffer systems, using the appropriate pK values as given by Bjerrum et al. (1957). Protease inhibitors were dissolved in potassium glutamate medium immediately before use; chymostatin was solubilized in DMSO (dimethyl sulphoxide), pepstatin in ethanol.

Cutaneous pectoris muscles were pinned out in Ringer’s solution containing 1·8mM-Ca and 5μgml⁻¹ A23187, which was initially solubilized in ethanol. Temperature, 20°C; pH7·l.

Fixation was by one of three methods: (1) Karnovsky fixative at 20°C for 30 min, with transfer to fresh fixative for a further 3–5 h, followed by two washes with 0·1 M-sodium cacodylate, pH 7·2, each of 30 min. The tissue was then cut into smaller pieces and postfixed in OsO ₄ for 2h at room temperature. The pieces of muscle were then cut into small blocks for washing in cacodylate buffer (two changes). (2) Glutaraldehyde (3 %) with a protocol as in method (1). (3) OsO ₄ (1 %) in venonal acetate buffer, pH 7·2 at 20°C for 3–5 h, followed by two washes in 0·1 M-sodium cacodylate, pH 7·2, each of 30 min. All preparations were dehydrated through a graded ethanol series, embedded in Spurr’s resin and sections were cut at 60–90 nm and stained with uranyl acetate and lead citrate.

At least three separate samples were taken from each muscle and the sections were scored blind on the electron microscope, by a colleague, for different types of damage; finally, the electron micrographs were again independently assessed.

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

This divalent cation ionophore caused characteristic and progressive damage in the intact cutaneous pectoris of R. temporaria over 10–30 min exposure. Fixation was with Karnovsky fixative. Initially, Z-line streaming was detected but, after some 20 min, breakdown of myofibrils occurred and some were contracted independently with Z-lines clearly out of register. After 20–30min 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 characteristic ultrastructural damage.

A total of 240 skinned cutaneous pectoris preparations were used in the present study and it was apparent that there were differences between R. pipiens and R. temporaria as well as marked variability between preparations (see below). Exposure to high saponin concentrations alone could promote ultrastructural damage and its effects were augmented by treatments that elevated intracellular [Ca]. Thus, the sucrose medium containing 0·5mM-EGTA (rather than the potassium glutamate) proved more satisfactory and the normal concentrations of saponin used were: R. pipiens 37·5 μgml⁻¹, 25min; R. temporaria 60 μg ml⁻¹, 30min. Electron micrographs (Fig. 1) of skinned preparations maintained for 90 min revealed a normal ultrastructure with clear myofilaments, sharp Z-lines in register, contracted mitochondria and with the SR not swollen.

Major changes resulting from damage could be readily detected with transmitted light and ×30 magnification in skinned preparations of the thin cutaneous pectoris muscle. Fixation with glutaraldehyde-based fixatives produced severe and visible damage within 60s of application and hence subsequently all fixation was with OsO₄.

Exposure of skinned fibres to potassium glutamate solutions in which Ca was buffered at 8×10⁻⁶M consistently produced damage, which was severe within 10 min in R. pipiens preparations and within 25 min in R. temporaria, and was readily detectable at ×30 magnification. Initially, this damage was seen as an area of contraction and abnormal folding of the muscle sheet, which gradually increased in area. Later, the contents in individual cells could be seen concentrated at specific foci and surrounded by clear cytoplasm.

Electron micrographs of Ca-damaged, skinned fibres revealed characteristic patterns of damage and, as with the intact cutaneous pectoris preparations, the results were invariably clear-cut. Whilst areas of undamaged myofibrils were found in all but the severest examples of damage, muscles in which the damage mechanism had been activated were immediately recognizable. The different types of degradative effect are listed in approximate order of severity in Table 1. Although individual sarcomeres appear to respond differently to the damage process (see individual myofibrils in Figs 3 and 14), there was a distinction, as with the results obtained with intact fibres, between sections of the muscle where damage was accompanied by extreme contraction (categories 6 and 7, Table 1), where the myofilament apparatus was clearly damaged but complete destruction did not occur during the 30-min exposure period (Fig. 11), and where hypercontraction did not occur and complete loss of actin and myosin filaments was found (Figs 4, 5, 19). Fig. 14 illustrates hypercontraction and complete dissolution in the same fibril. All these different types of damage were found in both species, but substantial differences could be found in different areas of the same muscle sheet; thus different categories of damage (together with apparently undamaged sections) could be found in one preparation and in adjacent sections of the same myofibril. Contraction-type damage (categories 6 and 7) appeared to be associated with the contractions observed with transmitted light. It is evident that the patterns of damage correspond with those caused by A23187 in intact fibres (Statham et al. 1976) whilst the hypercontraction 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 of skinned muscle fibres is identical with that found in intact fibres.

A series of experiments with both species showed that the effect of Ca was both time- and concentrationdependent. With the R. pipiens preparations, ultrastructural changes were not detected with [Ca] = 8×10⁻⁸M and developed only slowly in some preparations at [Ca] = 10⁻⁷M. In most preparations damage was detectable by eye in less than 10 min at 5 ×10⁻⁷ M-Ca and in all preparations in less than 5 min at 10⁻⁶M-Ca, and in less than 2 min at 8×10⁻⁶M. The R. temporaria preparation (summer frogs, skinned with 60 μg ml⁻¹ saponin), however, was consistently less sensitive and damage was not evident in the electron micrographs after 10 min exposure to 8×10⁻⁶M-Ca, but usually began after 18–20 min. Twenty to 25 min were required with 10⁻⁶ M and usually 30 min at 5×10⁻⁷M. It is noteworthy that at low [Ca] (<10⁻⁶M) the damage seen was more commonly in categories 1–5 but, nevertheless, contractiontype damage (categories 6 and 7) was also recorded and thus the same damage response was found across the Ca concentration range.

Some preparations showing category 5 damage (Table 1), where much of the contractile apparatus had been lost, revealed apparent thin, cytoskeletal, longitudinal elements linking the Z-lines, which appeared to be more resistant to the rapid degradative process than the myofilament apparatus. This characteristic effect was found in both intact (Fig. 20) and skinned (Figs 17–19, 21) amphibian muscle cells in which the degradative mechanism had been switched on in a variety of different ways. The appearance of these muscle cells closely resembled those of frog preparations in which the actin and myosin had been extracted with 0’6M-KI, so revealing the connectin (or titin/nebulin) network (Wang, 1982). This parallel elastic component (Maruyama et al. 1985) appears therefore, to be more resistant to the rapid degradative process than are the bulk myofibrillar proteins.

The sensitivity of some preparations to Ca and the variability between different muscles suggested that saponin might directly activate the damage process as well as permeabilize the sarcolemma. R. temporaria muscles were skinned for 30 min in a range of saponin concentrations, namely 40, 50, 55, 60, 75, 100, 120 and 150 μgml^−s, and Ca-induced damage was found to develop more rapidly with much more severe effects at the higher concentrations. At 150 μgml⁻¹, 8×10⁻⁶M-Ca damage could be detected in 90–120 s by transmitted light and, after 9 min, electron micrographs revealed almost complete dissolution of wide areas; a very much more dramatic response than with skinning with 60 μg ml⁻¹ saponin in this species. In one series of experiments, 150 μgml ⁻¹ saponin directly caused damage in R. temporaria.

Sr–EGTA buffered solutions also triggered identical patterns of damage in R. pipiens skinned preparations (Fig. 8), although substantially higher concentrations of free Sr were required. At 3×10⁻⁶M-Sr (25 min) individual damaged sarcomeres were detected together with some Z-line sliding; at 10⁻⁵M-Sr (after 45 min) the SR was additionally swollen in some areas; at 5×10⁻⁵M-Sr (25 min) substantial parts of the muscle remained undamaged but characteristic areas of myofilament breakdown were evident (category 1, Table 1). Finally, with 5×10⁻⁴M-Sr (25 min), clearly evident contractions and folding were seen under transmitted light and electron micrographs showed a full range of severe damage, including areas of extensive hypercontraction and mitochondrial multiplication (categories 6–9).

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

The skinned preparations were exposed to 8×10⁻⁶M-Ca with pH buffered at 7·3, 7·5 or 7#x00B7;6 (with the dissociation constant of the Ca-EGTA complex recalculated), but no protective effect was noted.

The R. pipiens skinned preparations were used for a range of experiments in which various protease inhibitors, both singly and in combination, were tested for possible effects in the reduction of damage. Table 2 summarizes the various protocols used and in the first series (experiments 1–6) the preparations were skinned for 30 min with saponin at 40 μg ml⁻¹, whereas in experiments 7 and 8 this procedure was increased to 40 min at 45 μg ml⁻¹ so as to increase the severity of skinning. In both series (experiments 1–8) the inhibitor was included in the skinning solution and in the subsequent rinse in the potassium glutamate medium and during the test exposure (25 min) to 8×10⁻⁶M-Ca, a concentration that consistently caused damage in control experiments. In experiment 9, the muscles were skinned in the absence of inhibitor and were then transferred to 10⁻³M-pCMB in 0·5mM-EGTA (i.e. zero [Ca]) and the preparation was fixed after only 5 min. The inhibitors were chosen to determine whether CANP or individual lysosomal cathepsins were itnplicated in rapid subcellular damage; combinations of inhibitors were selected to protect against possible lysosomal breakdown and the release of a range of acidic proteases.

In experiments 1–8, transfer to Ca caused rapid, severe and extensive damage, which was usually detectable by transmitted light within 10 min (Table 2). There were differences between preparations or between experiments in the time before changes were clearly visible, but no consistent protection was found in any of the experiments and electron micrographs prepared at the end of the experiment invariably revealed hypercontraction bands in addition to the other types of damage (Figs 5, 7, 11, 13). pCMB, an -SH blocker and hence an inhibitor of cysteine proteases, itself caused visible damage within 30 s in the absence of Ca, and electron micrographs of the preparation after only 5 min revealed widespread damage, both actin loss and disintegration of myofibrils (categories 1, 4 and 5) and also contraction damage (category 6). Thus, this damaged caused with zero [Ca] appears to be identical with the Ca-triggered damage.

One possibility concerning the action of Ca in triggering subcellular damage is that the ubiquitous calmodulin may be involved at one step in the degradative sequence and hence the anti-calmodulin drug TFP was tested on the saponin-skinned preparation. Three separate series of experiments with different batches of frogs (R. pipiens and R. temporaria) with appropriate protocols for skinning (R. temporaria = 60 (μg ml⁻¹ saponin, 30min; R. pipiens = 37·5 μgml⁻¹ saponin, 30 min) and with pre-exposure to TFP (10⁻⁵M or 3×10⁻⁵M) for 15 or 20min were used. Concentrations of TFP were chosen to replicate trials with TFP in intact muscle preparations (Duncan & Rudge, 1984). However, none of these measures provided any protection against the addition of 8×10⁻⁶M-Ca in the presence of TFP; both the time-course of the visual damage and the ultrastructural appearance (damage categories 1–8) after 30 min exposure were similar to those seen in the matched controls.

The saponin-skinned, cutaneous pectoris preparation (Endo & lino, 1980) proved to be a satisfactory system for determining the effect of Ca in initiating cellular damage, and in this thin sheet of muscle extreme damage can be detected visually by transmitted light and it has always been subsequently confirmed by electron microscopy. High concentrations of saponin appeared to be able to promote damage directly, suggesting that the system causing damage is membrane-bound and sensitive to membrane disruption. The action of glutaraldehyde-based fixatives in causing visible damage in skinned preparations almost immediately may also be via a membrane perturbation, as in its action on the Ca-ATPase of the SR (Thomas & Hidalgo, 1978).

The R. pipiens and R. temporaria preparations appeared to be genuinely different in their sensitivity to saponin and this observation may reflect a difference in the molecular structure of their muscle membranes. Furthermore, small, but consistent, differences in sensitivity between winter and summer frogs were also found. These poikilotherms have different optimal environmental temperatures and R. pipiens had been maintained at 23 °C, whereas A. temporaria was kept at 9°C, so that homeoviscous adaptation may well produce differences in the lipid composition of their membranes.

The results show unequivocally that intracellular Ca can trigger rapid damage in saponin-skinned amphibian skeletal muscle. The time taken to produce this damage is concentration-dependent and in both R. temporaria and R. pipiens the effective concentration range is probably about 10⁻⁷ to 10⁻⁵M-Ca, i.e. approximately the same range as the changes in concentration experienced in the cytosol during excitationcontraction-relaxation. As emphasized above, the saponin-skinned muscle may be partially activated; nevertheless the results do show that rapid damage can be triggered at very low cytosolic [Ca]. During contraction in the normal muscle cell the release of Ca will produce local, high levels of [Ca]₁, but these will be transient and the Ca²⁺ will be rapidly shuttled between the SR and the troponin C and parvalbumins.

The steady-state level of [Ca] ₁ in the muscle cell at rest (approximately 10⁻⁷ M) will be the result of passive influx and active efflux across the sarcolemma and of uptake by the SR (the low affinity mitochondrial Ca uptake is probably not implicated). At normal resting [Ca] ₁, in the closed system of the muscle cell, net active movement of Ca will be low, [Ca], being close to the lower sensitivity of the pumps for active transport. However, in the skinned muscle cell, immersed in a large volume of Ca–EGTA buffer, even at low free [Ca], active transport will continue as the pumps attempt the impossible task of reducing the free [Ca]. We conclude that it is not the concentration of Ca that is important in triggering myofilament damage, but rather the duration and magnitude of active movement of Ca across key membranes. In muscles where the steady-state level of [Ca] ₁ is elevated, perhaps quite modestly (e.g. in dystrophic muscle), damage may be triggered by the continued active transport of Ca over a period of many minutes.

Damage can be detected visually after 90 s in skinned preparations at 8×10⁻⁶M-Ca and this rapidity is consistent with results obtained with rat hearts in Ca-paradox experiments. Return of extracellular Ca after Ca-free perfusion caused the typical loss of cellular proteins but, significantly, if the hearts were repleted with Ca for only 30 s and then returned to Ca-free perfusion, the resultant protein loss was the same (Hunt & Willis, 1985).

The action of Sr–EGTA in triggering damage, albeit at higher concentrations (effective range 3×10⁻⁶M to 5×10⁻⁴M), is in accord with observations on intact preparations, particularly amphibian and mammalian hearts where Sr can substitute for Ca in triggering damage (DeLeiris & Feuvray, 1973; Goshima et al. 1978) that was ultrastructurally identical (Rudge, 1983).

The damage produced in the skinned cells is summarized in Table 1 and can be classified into two different types of response. First, loss of actin filaments, breaking and disintegration of the myofibrils, and myofilaments separating from the Z-line (categories 1, 3 and 4); this type of damage is characterized by sharp and clearly defined Z-lines. Second, categories 6 and 7 appear to accompany contraction of the myofilament apparatus and concomitantly the Z-line widens and becomes blurred (see Figs 10, 11, 12, 17), particularly in areas of hypercontraction. In areas of hypercontraction, the myofilament apparatus appears to be protected from disassembly and thus a sarcomere may respond with one of the two types of damage. The patterns of damage found in skinned cells closely resemble those found in intact cutaneous pectoris treated with A23187 (Statham et al. 1976). Hypercontraction bands and dramatic mitochondrial subdivision are also characteristically found in damaged amphibian and mammalian cardiac muscle and in mammalian skeletal muscle in which [Ca]₁ has been raised (Publi-cover et al. 1978; Duncan & Smith, 1980; Duncan et al. 1980; Rudge & Duncan, 1984a,b).

Thus, we conclude (1) that the characteristic and common patterns of rapid damage (both in vivo and in vitro) in vertebrate skeletal and cardiac muscle reflect a common underlying sequence of cellular events that culminate in specific ultrastructural degradation of the myofilaments; (2) that the damage caused by A23187 in intact amphibian (Statham et al. 1976) and mammalian (Publicover et al. 1978) skeletal and cardiac (Rudge & Duncan, 1980, 1984a) muscles are indeed triggered by rises in [Ca] ₁ and in Ca fluxes in the cell.

Ruthenium Red causes rapid and characteristic damage in intact cardiac and skeletal muscles (Duncan et al. 1980; Rudge & Duncan, 1980, 1984a), but was ineffective in the skinned preparation. We conclude that in the intact cell Ruthenium Red inhibits the Ca-ATPases of the Ca pumps of the sarcolemma, SR or mitochondria and thereby raises [Ca] ₁ and so, in turn, initiates myofilament damage.

The experiments with pCMB in the presence of EGTA, which cause the same characteristic myofilament damage (presumably via an action on key -SH groups), demonstrate that an alteration in [Ca] _i is not the only means whereby the underlying cellular mechanism can be initiated.

Previous in vitro experiments with trifluoperazine (TFP) and intact skeletal and cardiac muscles showed that not only did this anti-calmodulin agent fail to protect against the characteristic ultrastructural damage produced by a variety of different means, but that exposure to 10⁻⁵M-TFP alone produced identical patterns of damage (Duncan & Rudge, 1984). Clearly, calmodulin could act to modify either the systems regulating [Ca], or the Ca-triggered damage process itself. Since TFP does not protect against Ca-induced damage in the skinned cell in the present experiments, we conclude that calmodulin does not have an overriding control of the rapid damage process that proceeds in the presence of TFP. Presumably, TFP in vitro inhibits the calmodulin-activation of the Ca-ATPase of the sarcolemma thereby raising [Ca] _i.

Previously, muscle damage has been measured either qualitatively by histological or ultrastructural assessment, or quantitatively by assaying the release of cytoplasmic proteins, a method that reflects only the lack of integrity of the sarcolemma. We do not yet know the sequence of events in cellular damage, but recent studies (Duncan & Jackson, 1987) have shown that at least two independent pathways are involved: (1) PLA₂ (phospholipase A₂) activation and subsequent lipoxygenase activity culminating in sarcolemma damage; and (2) a separate system that produces the characteristic destruction of the myofilament apparatus.

A number of theories are extant concerning the mechanisms underlying cellular damage but the present studies with skinned fibres show that some of these are not implicated in the specific and rapid damage of the myofilaments and these are discussed below.

pH₁ falls markedly in malignant hyperthermia and in cardiac ischaemia, with irreversible cell injury occurring at pH 6·3 (Gebert et al. 1971; Ichihara et al. 1979; Schaper & Knoll, 1979). It is proposed that protons are emitted in exchange for Ca uptake by the SR and mitochondria, that acidification also results from ATP breakdown (Grinwald & Nayler, 1981) and that the resulting acidosis injures cells and causes membrane damage (Ganóte, 1983). However, ‘intracellular’ pH is firmly buffered in the skinned cells and no protective effect was found even when the pH was raised to 7·3, 7·5 or 7·6, supporting the results of studies with intact muscle cells showing that rapid damage is triggered even when the mitochondria are uncoupled (Duncan et al. 1980).

During rapid damage, in both skeletal (in malignant hyperthermia) and cardiac (in ischaemia; Ganóte, 1983) muscles, the levels of high-energy phosphates fall dramatically. It is proposed that this process directly causes damage and rigor complexes and also permits alterations in membrane permeability (Jennings & Reimer, 1981) or changes in electrolyte levels (Nayler, 1981) to occur.

However, rapid damage in less than 120 s was triggered by Ca in the skinned muscle with 3 mM-ATP and it is unlikely that high-energy phosphate depletion is the direct cause of myofilament damage.

Since CANP was localized in skeletal and cardiac muscles, considerable work has been directed towards elucidating its possible role in myofibrillar degradation. CANP is also activated by Sr and its properties and role in degradation 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 prevented rapid Ca-activated damage or pCMB-induced damage in skinned muscle preparations (Table 2), confirming previous conclusions from studies with intact skeletal (Duncan et al. 1979) and cardiac (Rudge & Duncan, 1984a) muscle that CANP is not the major element in rapid cellular damage.

Titin and nebu-lin (or connectin) form an elastic network within muscle cells and both are rapidly cleaved by endogenous and exogenously added proteases, especially CANP (Wang, 1982). If the cytoskeletal remnants left after rapid cellular damage (Figs 17–21) are indeed a resistant titin–nebulin network, these observations provide confirmatory evidence that CANP is not involved. It is thus also unlikely that lysosomal proteases (see below) are responsible for rapid myofilament degradation and it seems that this damage, stimulated by pCMB, may be a preferential attack on -SH-linked subunits, since titin and nebulin are believed to be constructed from (non-disulphide) covalently linked subunits (Wang, 1982).

Since lysosomal cathepsins B and D degrade myofibrillar proteins (Schwartz & Bird, 1977; Matsumoto et al. 1983), a number of workers have suggested that cellular degradation is produced by the release of these enzymes (Kennett & Weglicki, 1978; Wildenthal & Crie, 1980; Katanuma & Noda, 1982), and the fall in pH_i (see above) would permit their activity once free in the cytosol. Lysosomal enzyme activity has been reported to be elevated in various muscular dystrophies (Pearson & Kar, 1979; Noda et al. 1981) and in ischaemic heart (Wildenthal, 1978).

Studies with protease inhibitors on intact muscle are bedevilled by uncertainty as to whether they can penetrate the sarcolemma, but none of the inhibitors tested on skinned muscles in this study prevented rapid myofilament damage, nor were any differences from normal, characteristic damage noted, even when the skinning procedure was extended (Expts 7 and 8, Table 2). Cathepsin D is inhibited by pepstatin and chymo-statins; cathepsins B, H and L are inhibited by thiol-blocking agents, iodoacetate and leupeptin. The mixtures of inhibitors (Expts 5–7, Table 2) were chosen to protect against the release of mixed lysosomal proteases. It is also unlikely that acidic proteases could achieve complete degradation in 90 s at pH 7·5, and it is evident that lyososmal cathepsins are not involved in rapid myofilament degradation in amphibian skeletal muscle.

To conclude, low levels of Ca trigger rapid and characteristic myofilament damage in skinned skeletal muscle cells. Neither a fall in pH₁, a fall in high-energy phosphate reserves, activation of CANP nor the release of lysosomal enzymes is directly involved, although all may have a peripheral role in vivo.

The assistance of Mr J. L. Smith with the electron microscopy and of Miss S. Scott in preparing the manuscript is gratefully acknowledged.