Inhibition of lysosomal function in red and white skeletal muscles by chloroquine

EXPERIMENTAL

NEUROLOGY

Inhibition

of Lysosomal Function in Red and White Skeletal Muscles by Chloroquine

W. T. STAUBER, Department

71, 295-306 (1981)

A. M. HEDGE,

J. J. TROUT,

AND B. A. SCHOTTELIUS'

of Physiology, West Virginia University Medical Center, Morgantown, West Virginia 26506 and Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242 Received

March

IO, 1980; revision

received

July

9, 1980

In chickens treated 7 days with chloroquine, morphological observations and chemical analyses were in agreement with our hypothesis that lysosomes degrade some fraction of skeletal muscle mitochondria, plasma membrane, and glycogen but apparently not normal myofibrillar proteins. After chloroquine inhibition of lysosomal digestion, autophagy was apparent in anterior (ALD) and posterior (PLD) latissimus dorsi muscles. Membrane-limited vacuoles (autophagic vacuoles) contained mitochondria, membranes, and glycogen. To help identify the vacuolar constituents, a variety of marker enzyme activities and chemical analyses were monitored for the ALD and PLD muscles. Parallel increases in 5’-nucleotidase and cytochrome oxidase specific activities correlated with the increase in observable inciusions. Autolytic rates were increased in acidic (pH 4.0) but not in alkaline (pH 8.5) or neutral (pH 7.0) conditions when homogenates were prepared from muscles of chloroquine-treated chickens. The removal of chloroquine inhibition in vitro by the preparation of tissue homogenates resulted in increased proteolysis by lysosomal enzymes presumably due to an increase in available substrates derived from mitochondria and plasma membranes.

INTRODUCTION Lysosomes have been associated with diseases such as denervation atrophy (13) and muscular dystrophy (16,23,33) as well as with starvation Abbreviations: ALD, PLD-anterior, posterior latissimus dorsi; ATP-adenosine triphosphate; IMP-inosine monosphosphate; TCA-trichloroacetic acid. r This work was supported in part by a grant from the American Heart Association with local support from the Iowa Heart Association to B.A.S., by U.S. Public Health Service grant HL 25388 to W.T.S., and by a program project grant HL-14388 to the Cardiovascular Center at the University of Iowa. J.J.T. is a National Institutes of Health (NINCDS) postdoctoral fellow. 295 0014-4886/81/020295-12$02.00/O Copyright All rights

8 1981 by Academic Press, Inc. of reproduction in any form reserved.

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(2). Increased numbers of lysosomes as visualized by histochemical techniques, increased specific activities of lysosomal enzymes, and extrapolations to other tissues such as liver constitute the substance of the evidence. However, white (phasic) skeletal muscles, which have been shown to contain low lysosomal enzyme activity (24, 30) and few morphologic equivalents of lysosomes (35), are most commonly atrophied in these conditions. Chloroquine-induced myopathy represents one exception to this general scheme in that red (tonic) muscles are predominantly involved (20). Chloroquine accumulates in lysosomes and inhibits lysosomal digestion by elevating intralysosomalpH (7). Chloroquine also specifically inhibits the lysosomal proteinase, cathepsin B (36). Therefore, the red muscle involvement observed in chloroquine-induced myopathies is consistent with the concept of a more important role of lysosomes and lysosomal proteinases in red rather than white muscle types. This study was designed to identify which intracellular components might be digested by the vacuolar apparatus of muscle cells. The overall hypothesis was that if degradation or lysosomal processing could be slowed by chloroquine treatment, accumulations of segregated material destined to be digested by muscle cell lysosomes would arise. The results indicate that autophagy of skeletal muscle constituents may be limited to mitochondria and plasma membranes and some fraction of cellular glycogen. MATERIALS

AND METHODS

Cornish-cross chickens at least 2 weeks old received 25-mg/kg i.p. injections of chloroquine for 7 days and were killed by decapitation on the 8th day. The anterior (ALD) and posterior (PLD) latissimus dorsi muscles were removed, cleaned of fat and connective tissue, and weighed. The muscles were twice homogenized 5 s in a VirTis 45 blender at top speed and subsequently rehomogenized with a glass homogenizer in an ice-slurry. For glycogen and RNA determinations, the homogenization solution was 0.1 M acetate buffer, pH 5.0; for autolysis studies, distilled water; and for enzyme analyses, 0.25 M sucrose, pH 7.2. Similarly, homogenates from ALD and PLD muscles and liver were obtained from control animals of the same age. RNA content was determined by the orcinol method of Schneider (26) and glycogen content by the amyloglucosidase technique of Keppler and Decker (17). Protein content was approximated by the method of Lowry et al. (19) using human serum albumin as the standard. Phosphate was determined by the sensitive technique of Marinetti et al. (21).

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Aldolase was determined using Sigma Kit No. 750 (Sigma Chemical Co.). Cathepsin D, cytochrome oxidase, and /+glucuronidase were measured as described for muscle homogenates (4); cathepsin B by the method of Barrett (1); and K-ATPase, which is specific for sarcoplasmic reticulum when basal Mg-ATPase has been subtracted, was assayed by the method of Duggan (8). Alpha-glucosidase and N-acetyl+-glucosaminidase were assayed as described elsewhere for skeletal muscle (30). 5’-Nucleotidase was determined by the method of Headon et al. (14) using 5’-inosine monophosphate (IMP) at pH 6.5 which is specific for muscle. Acid phosphatase was monitored using 0.05 M P-glycerophosphate in 0.10 M acetate buffer, pH 5.0. Myosin-ATPase was determined by measuring the phosphate released from adenosine triphosphate (ATP) in solution of 50 mM Tris, 5 mM EDTA, 400 mM KCl, and 5 mM Tris-ATP,pH 7.5, after membranes were disrupted using Triton X-100 (TVB solution). TVB solution (0.01% (w/v) Triton X-100, 1 mM EDTA, and 1 mM sodium bicarbonate) was used for dilutions of homogenates for lysosomal enzyme determinations, cytochrome oxidase, and myosin-ATPase assays. For electron microscopy both bilaterally situated anterior latissimus dorsi (ALD) muscles were fixed 5 min in situ and by immersion for an additional 1 h in 2.5% glutaraldehyde in 0.2 M cacodylate-HCl buffer,pH 7.3; diced into l-mm cubes; rinsed overnight in 0.2 M cacodylate-HCl buffer, pH 7.3; and processed for morphological studies as described elsewhere (34). All samples were embedded in Epon 812. Ultrathin sections were cut with a diamond knife, mounted on naked 200-mesh copper grids, and after staining with uranyl acetate and lead citrate viewed in a Hitachi HU-125-E-1 electron microscope operated at 75 kV. Complete comparative studies of the morphological observations in ALD and PLD muscles, which further substantiate the results presented in Fig. 3, will be published later. The experimental protocol is based on the hypothesis that chloroquine inhibits lysosomal processing of proteins (36). Because skeletal muscle cells do not actively phagocytose macromolecules, the most likely route for macromolecules to arrive within the lysosome is by autophagy. Autophagy is defined as the segregation and digestion of cellular constitutents (9) and is represented by the following scheme: Autophagy Step 1: Segregation of cellular constituents by intracellular membranes (autophagosomes) Step 2: Fusion of segregated material with enzyme-laden lysosomes (primary lysosomes) Step 3: Digestion within lysosomes (secondary lysosomes)

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ET AL.

CYTOCHROME

0

100 GLYCOGEN

200

ONDASE

100

0 CYTOCHROME

20 0 OXIDASE

FIG. 1. Lysosomal enzyme activity as a function of substrate concentration in control anterior (0) and posterior (A) latissimus dorsi muscles and control liver (x). Left-aglucosidase and glycogen; right top-cathepsin B and a marker protein; right lower-cathepsin D and a marker protein. Cytochrome oxidase activity represents the marker protein. Nonspecific units of activity.

Chloroquine was documented to inhibit protein degradation at step 3 because it can directly inhibit the lysosomal protease, cathepsin B (36). It is possible that step 2 also is inhibited due to drug-induced changes in membrane lipids or elevations in intralysosomal pH (7). If step 1 is not altered or is altered to a lesser extent, then increased numbers of autophagic vacuoles should obtain as reported by others for skeletal muscle following chloroquine (20). When the tissue is homogenized and the inhibitor, chloroquine, is diluted by the homogenization media, the segregated material would be available for enzymatic digestion. Thus, increased autolytic rates would result because of an increase in natural substrates. For such autolysis studies, homogenates were incubated 3 h in buffers, the reaction was stopped with 10% trichloroacetic acid (TCA), and the TCA-soluble products were determined as described elsewhere (29). Enzymes inside autophagosomes also might be expected to retain some enzymatic activity because of elevated intravacuolar pH which results from the protonation of chloroquine (7). Thus, increased marker enzyme activity, which correlated with the increased autolytic rates, might identify the cellular components destined for lysosomal degradation.

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RESULTS Certain lysosomal enzyme activities (proteases and glucosidase) were found to correlate with cellular constituents (specific enzymes and glycogen) which, by their chemical nature, might serve as substrates for these enzymes (Fig. 1). The specific activity of a-glucosidase was found to parallel tissue glycogen content in avian liver and ALD and PLD muscles from control untreated chickens. Similarly, both cathepsin B and D activities varied with a specific protein, cytochrome oxidase, in control ALD and PLD muscles. In contrast, only cathepsin B and not cathepsin D, activity was found to parallel another protein, 5’-nucleotidase (Fig. 2), in control muscles. These data imply that at least some fraction of cellular glycogen, mitochondria (as indicated by cytochrome oxidase activity), and plasma membrane (as indicated by 5’-nucleotidase activity) might be normally degraded by skeletal muscle lysosomal enzymes during autophagy. Thus, inhibition of lysosome digestion by chloroquine would be expected to result in accumulations of some portion of plasma membrane, and glycogen normally processed by lysosomes. In 7-day chloroquine-treated chickens, morphological observations of the ALD and PLD muscles were in agreement with the overall hypothesis 200 r=.5398

.

20 5’-NUCLEOTI

30 DASE

2.0 r=.899

20 5’-NUCLEOTIDASE

30

FIG. 2. F’roteinase activity as a function of a marker protein (5’-nucleotidase activity). (0) Control anterior latissimus dorsi muscles; (A) posterior latissimus dorsi muscles. Nonspecific units of activity.

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FIG. 3. Autophagy in anterior latissimus dorsi muscle after 7 days of chloroquine treatment. Membranes shown in the process of segregating an area of cytoplasm. Insets show two autophagic vacuoles. Stained with uranyl acetate and lead citrate, x36.800; upper left inset, ~42,400; lower right inset, x48,800.

that chloroquine inhibited the digestive step in autophagy (see Materials and Methods). Autophagy was very apparent (Fig. 3) with mitochrondria, membranes, and glycogen present inside membrane-limited vacuoles. The most prevalent vacuoles were membrane-limited mitochondria and myelin figures (membranous inclusions) and the least frequently observed were glycogen accumulations. A variety of enzymes and tissue components of muscle cells were compared in ALD and PLD muscles, both control and after 7 days of chloroquine treatment (Table l), to help identify which cellular constituents had accumulated. Aldolase and myosin-ATPase activity, representative of cytoplasmic proteins, and K-ATPase activity, indicative of a sarcoplasmic reticular membrane protein, did not change after chloroquine treatment. In contrast, cytochrome oxidase activity and 5’-nucleotidase activity increased in muscles from chloroquine-treated chickens compared with controls. The five lysosomal enzymes measured

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MUSCLE

(cathepsin D, cu-glucosidase, N-acetyl-P-glucosaminidase, P-glucuronidase, and acid phosphatase) did not significantly vary from controls. Cathepsin B was not determined because chloroquine is known to inhibit its activity (34). RNA content decreased for the ALD and not the PLD muscles and glycogen content did not change significantly in either muscle. To determine if the in viva inhibition of digestion by chloroquine would indeed lead to an increase in putative substrates for lysosomal digestion such as mitochondrial and plasma membrane proteins, autolysis experiments were carried out at threepH values for which proteolytic activity has been described (Table 2). Chloroquine’s inhibitory effect would be minimized by dilution (see Materials and Methods) and thus allow degradation to proceed at increased rates due to an increase in substrate concentration. An almost twofold increase in TCA-soluble products was noted at pH 4.0 but no changes were detectable at pH 7.0 and 8.5. The release of hydrolysis products at acid pH (pH 4.0) was found to correlate TABLE

1

Enzyme Specific Activities, Glycogen, and RNA Content for Anterior (ALD) and Posterior Latissimus Dorsi (PLD) Muscles from Chickens Treated 7 Days with Chloroquine” ALD Control Aldolaseb Myosin-ATPase’ K-ATPase’ Cytochrome oxidase” 5’-Nucleotidase’ RNA’ Glycogen’ a-Glucosidase’ /Xllucuronidase’ Acid phosphatase’ N-Acetyl-Pglucosaminidase Cathepsin DC Cathepsin BC

PLD Chloroquine

Control

Chloroquine

17.4 58.2 13.6

k 0.5 -e 2.7 ? 1.2

16.5 52.7 14.1

2 1.6 + 5.6 * 0.7

38.1 79.0 74.8

” 6.0 k 4.2 2 5.8

39.9 k 1.8 85.1 * 5.2 83.2 + 3.1

37.0 0.81 23.4 12.5 0.058 0.221 0.95

k 2.3 2 0.13 + 1.0 f 3.0 k 0.003 % 0.010 + 0.02

60.2 1.29 18.0 8.7 0.055 0.219 1.01

2 + * 2 2 -t f

16.7 0.64 14.2 20.3 0.118 0.178 0.58

+ k ? + + 2 k

26.6 1.01 14.1 13.4 0.129 0.192 0.60

0.30 k 0.01 1.91 2 0.07 0.038 k 0.004

D Numbers indicate the mean 2 b Unitslmg protein. c munits/mg protein. rl munits/mg protein (6). c &mg protein. f Not measured. * P < 0.05.

SE

2.2* 0.08* 1.2* 1.0 0.002 0.021 0.03

0.31 2 0.02 2.04 t 0.07 +’

0.6 0.11 0.4 4.8 0.003 0.012 0.01

0.14 t 0.01 0.92 + 0.03 0.022 * 0.003

of six muscles assayed in duplicate.

+ 2 + t ? ” +

2.2* 0.07* 1.0 4.5 0.003 0.013 0.01

0.14 2 0.01 1.00 -c 0.04 +’

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TABLE

2

Autolytic Activity for Anterior (ALD) and Posterior (PLD) Latissimus Muscles from Chickens Treated 7 Days with Chloroquine” pH 4.0

pH 7.0

pH 8.5

7.2 + 0.6 7.6 k 1.1

4.8 + 0.5 3.7 + 0.7

4.0 2 0.7 5.3 + 1.1

4.0 + 0.7 5.2 4 0.7

Autolysis (PLD) Control Chloroquine

18.5 2 2.5 33.5 r 3.0* Autolysis (ALD)

Control Chloroquine

25.8 + 1.8 44.2 2 4.8*

’ Numbers indicate mean + protein. * P < 0.05.

SE

for 10 muscles each assayed in triplicate in punitsimg

with changes in both cytochrome oxidase activity (mitochondrial content) and 5’-nucleotidase activity (plasma membrane content) (Fig. 4). DISCUSSION The question of the function of lysosomes in muscle biology and pathology remains to be answered. Although lysosomal cathepsins of skeletal muscle are capable of degrading actin and myosin (27), only phagocytic cells (25) and fetal myocytes (32) have been observed to contain recognizable contractile proteins (myofibrils) inside digestive vacuoles (lysosomes). With the exception of chemically altered actinomyosin, i.e., abnormal proteins (lo), only mitochrondria, membranes, and glycogen have been identified inside lysosomes of muscle cells under most conditions (5, 18). Based on similar observations, Stauber and Schottelius (31) proposed that lysosomes normally function in autophagy of nonmyofibrillar components except abnormal actinomyosins or error proteins referred to above. Comparative studies of anterior (ALD) and posterior (PLD) latissimus dorsi muscle lysosomes indicated that all lysosomal enzymes tested except a-glucosidase were enriched in an oxidative muscle, the tonic ALD. The high specific activity of a-glucosidase in a glycolytic muscle, the phasic PLD, (Table 1) confirms earlier work on lobster tail muscle in which even greater activity was observed (28). In contrast, after denervation of the PLD muscle, all lysosomal enzymes tested increased in specific activity except a-glucosidase which decreased (31). Similarly, glycogen content was reported to decrease after denervation (15). These experiments

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suggested that variations in muscle glycogen content were paralleled by proportional variations in cu-glucosidase activity. As glycogen is not present in circulating fluids because of the presence of plasma amylase, the only known pathway for glycogen to enter the vacuolar apparatus is by autophagy and this is true for most cells. Thus the correlation is this study between cu-glucosidaseactivity and glycogen content in two muscle types and liver would support the hypothesis that lysosomal enzyme activity was proportional to substrate content. It was reasonable to believe that if lysosomal digestion were inhibited, material normally degraded by lysosomes would accumulate proportion-

CYTOCHROME

01

OXIDASE

15 30 5’-NUCLEOTIDASE

45

FIG. 4. Autolytic activity as a function of two marker proteins (cytochrome oxidase and 5’nucleotidase, respectively); nonspecific units of activity. (0) Anterior latissimus dorsi (ALD) muscles from control chickens; (0) ALD muscles from 7-day chloroquine-treated chickens; (A) posterior latissimus dorsi (PLD) muscles from control chickens, (A) PLD muscles from 7-day chloroquine-treated chickens.

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ally to the inhibitory effect on the enzyme(s) responsible for their degradation. Because chloroquine is a known inhibitor of cathepsin B. the accumulation of cathepsin B substrates (proteins) should predominate. Indeed, cytochrome oxidase and 5’-nucleotidase content in muscle increased after short-term chloroquine treatment consistent with an inhibition of cathepsin B. If cytochrome oxidase and 5’-nucleotidase are markers of substrates for lysosomal cathepsins and the increases in their activity resulted from decreased degradation, autolytic activity should also increase when the inhibition by chloroquine is removed (See Materials and Methods). Such increased autolytic rates (at acid pH values) were observed when muscle homogenates were tested (Fig. 4). These increased rates correlated with variations in 5’-nucleotidase and cytochrome oxidase activities. In fact, the autolytic rates and cathepsin B specific activities were of the same order of magnitude (micromoles of product released). Consequently, it would appear that some portion of the mitochondria and plasma membrane is degraded by lysosomal cathepsins, predominantly cathepsin B. It is possible that components which enter muscle lysosomes at slow rates were not observed because of the limited time span studied (7 days). However, even after 42 days of chloroquine treatment, little morphologic variation in the segregated material was observed (20). Lockshin and Beaulaton (18) also reported that mitochondria and ribosomes, but not myofibrillar constitutents, were observed in autophagic vacuoles during intersegmental muscle degeneration. Their concept of a highly selective role for lysosomes in muscle tissues is consistent with the data reported by us in this paper and elsewhere (31). It is generally accepted that muscle atrophy results from increased proteolysis and not decreased synthesis of contractile proteins ( 11,12). The presence in skeletal muscle of a full complement of lysosomal proteinases led many investigators to implicate lysosomes and lysosomal proteinases in the autophagic degradation of normal proteins (3) similar to that seen in liver treated with glucagon or in the absence of insulin (22). Inhibitors of proteinase activity, such as chloroquine, have helped unravel the role of various enzymes in the turnover qf proteins in a variety of cells, but because of its effect on intralysosomal pH, one cannot rule out other mechanisms of chloroquine action such as prevention of delivery of enzymes to lysosomes, or inhibition of fusion between enzyme-containing vesicles and autophagic vacuoles. An understanding of lysosomal proteinase function in muscle cells may be forthcoming by the application of proteinase inhibitors that are even more specific than chloroquine. However, from the data at hand, increased specific activities of lysosomal proteolytic enzymes that accompany muscle diseases do not in themselves implicate lysosomes in the

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degenerative process and indicate the need for antilysosomal agents. Indeed, caution probably should be exercised in the administration of proteinase inhibitors directed against lysosomal proteinases, to dystrophic animals in attempts to retard degeneration, because, like chloroquine, these inhibitors may induce a new muscle disease. REFERENCES 1. BA&TT, Biochem.

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