Different

Mechanisms

Marc E. Tischler,

of Increased Proteolysis in Atrophy Induced or Unweighting of Rat Soleus Muscle

Sara Rosenberg,

Soisungwan

Satarug,

Erik J. Henriksen,

Christopher

by Denervation

R. Kirby, Margaret

Tome,

and Peter Chase Mechanisms of accelerated proteolysis were compared in denervated and unweighted (by tail-cast suspension) soleus muscles. In vitro and in vivo proteolysis were more rapid and lysosomal latency was lower in denervated than in unweighted muscle. In vitro, lysosomotropic agents leg, chloroquine, methylamine) did not lessen the increase in proteolysis caused by unweighting, but abolished the difference in proteolysis between denervated and unweighted muscle. Leucine methylester, an indicator of lysosome fragility, lowered latency more in denervated than in unweighted muscle. 3-Methyladenine, which inhibits phagosome formation, increased latency similarly in all muscles tested. Mersalyl, a thiol protease inhibitor, and 8-(diethylamino)octyI-3,4,5-trimethoxybenzoate hydrochloride (TM&8). which antagonizes sarcoplasmic reticulum release reduced accelerated proteolysis caused by unweighting without diminishing the faster proteolysis due to of Ca”, denervation. Calcium ionophore (A231871 increased proteolysis more so in unweighted than control muscles whether or not Ca’+ was present. Different mechanisms of accelerated proteolysis were studied further by treating muscles in vivo for 24 hours with chloroquine or mersalyl. Chloroquine diminished atrophy of the denervated but not the unweighted muscle, whereas mersalyl prevented atrophy of the unweighted but not of the denervated muscle, both by inhibiting in vivo proteolysis. These results suggest that (I I atrophy of denervated, but not of unweighted, soleus muscle involves increased lysosomal proteolysis, possibly caused by greater permeability of the lysosome. and (2) cytosolic proteolysis is important in unweighting atrophy, involving some role of Ca2+ -dependent proteolysis and/or thiol proteases. 0 1990 by W. B. Saunders Company.

0

UR KNOWLEDGE of the mechanisms of atrophy in various types of muscle wasting remains limited. In show that denervated2,3 vitrole4 and in vivo3-6 measurements and unweighted’,5,6 muscles lose protein as a consequence of decreased protein synthesis and accelerated protein degradation. Glucocorticoids have been considered as a possible contributing factor to atrophy of these muscles. Although denervated muscles seem more susceptible to cortisoneinduced atrophy,* denervation atrophy likely is not caused by enhanced sensitivity of the muscle to these hormones.’ Unweighting atrophy also is apparently not a consequence of cortisol effects,* despite increased levels of cytosolic glucocorticoid receptors in the unweighted muscle.’ Insulin also is an important regulator of muscle protein metabolism. Some responses to insulin in unweighted soleus muscle”,” differ considerably from those reported for denervated skeletal muscles.‘2-‘5 While denervated muscles show

From the Departments of Biochemistry and Physiology, University of Arizona Health Sciences Center, Tucson. AZ. Supported in part by a National Aeronautics and Space Administration (NASA) Grant No. NAG2-384, by NASA Graduate Researcher Program Fellowships to E.J.H. and C.R. K.. by National institutes of Health Training Grants No. NS-07309 and HL-07249 to the Department of Physiology, and by Biomedical Research Support Grant No. RR-05675 to the College of Medicine. Preliminary aspects of this work were presented at the Second International Symposium on Proteases and was reported in the Proceedings from the Symposium which appeared in Advances in Experimental Medicine and Biology 40:23.5-242, 1989. Present addresses: S. Satarug, Department of Biochemistry, Faculty of Medicine, Khon-Kaen University, Khon-Kaen, Thailand; E.J. Henriksen. Department of Internal Medicine, Washington University School of Medicine, St Louis, MO. Address reprint requests to Marc E. Tischler, PhD. Department of Biochemistry, University of Arizona, Tucson, AZ 85724. D 1990 by W.B. Saunders Company. 0026-0495/90/3907-0016$03.00/0 756

insulin resistance for glucose metabolism, unweighting the soleus muscle induces an increased response of glucose metabolism to insulin at the time atrophy becomes apparent.“,” Similarly, insulin produces greater anabolic effects on protein metabolism in unweighted than in denervated muscle.16 To examine further the differences in insulin response of denervated and unweighted muscles, investigators have measured the affinity and capacity of these muscles for binding insulin. Neither intervention affects the affinity of the receptor for insulin.‘0,‘3,‘4 In the denervated muscle the binding capacity is also unaffected. Therefore, the insulin resistance of this tissue must be a consequence of a postreceptor defect causing altered receptor-function coupling.‘3,‘4 In contrast, unweighted soleus muscle shows an increased binding capacity for insulin per tissue mass due to no apparent decrease in total receptor number during muscle atrophy.” These findings point to fundamental differences between denervated and unweighted muscle other than the distinction of innervation status. In this report, further study of these dissimilarities is considered by comparing mechanisms of proteolysis in unweighted and denervated soleus muscles. Muscle contains proteolytic pathways in both the lysosomes and cytosol.“,” The relative contributions of these two pathways in the degradation of certain groups of proteins is known. For instance, membrane proteins, such as the insulin receptor, are degraded primarily in the lysosome.‘9-23 Because insulin binding studies suggest that the insulin receptor undergoes net breakdown in denervated but not in unweighted muscle, we hypothesize that lysosomal proteolysis may be more important in denervation than in unweighting atrophy. In this study, we focused on the possibility of different sites of accelerated proteolysis in denervated and unweighted muscle. Several lysosomotropic agents were used to test whether lysosomal proteolysis might be enhanced in denervated muscle, relative to unweighted muscle, by measuring in Metabolism, Vol 39, No 7 (July), 1990:

pp 756-763

PROTEOLYSIS IN ATROPHYING

MUSCLE

protein degradation and latency of lysosomal protease activit,y. Effects of other agents were tested to further characterize proteolysis in the atrophying muscles. These agents included mersalyl, an inhibitor of thiol proteases24.25 (eg, Cal+-activated protease), and g-(diethylamino)octyl3,4.5-trimethoxybenzoate hydrochloride (TMB-8), an antagonist of calcium release from sarcoplasmic reticulum.‘b Finally, we have tested the effects of chloroquine and mersalyl in vivo to determine the possible physiological significance of our hypothesis. vitro

MATERIALS

AND

Frozen muscles were homogenized in 2.5 mL of ice-cold 2% perchloric acid. The protein pellet was washed with 5 mL of this acid and resuspended in 6 mL of 0.3 N NaOH. A I-mL aliquot of this solubilized protein was analyzed for protein by the Lowry procedure using bovine serum albumin as a standard.‘O A 0.5-mL intracellular or 0.75-mL protein hydrolysate aliquot was added to 5 mL of ACS to determine the radioactivity in these fractions. The fractional rate of synthesis was calculated as (Q/(0.9 . S, . t).‘9 Growth rate was estimated as the difference in the average protein content of muscles from injected animals and of muscles taken from parallel groups of animals killed 24 hours earlier. Corrections were made for differences in initial body weights between animals.”

METHODS

Treatment of Animals and 100 to 115 g for in vivo studies; Sasco Sprague-Dawley, Omaha, NE or Harlan SpragueDawley, Madison WI) were maintained on food and water ad libitum and treated as described below. Unweighted animals were tail-casted and suspended in a head-down position so that the hind legs were elevated above the floor of the cage for the period indicated.’ Tail casts consisted of Hexcelite orthopedic tape (Kirschner Medical, Timonium, MD) and medical grade elastomer (Factor II, Lakeside, AZ). Control animals also were tail-casted, but remained weight-bearing. For denervating the soleus muscle, a 3- to 5-mm section of the sciatic nerve was removed. All manipulations of the animals were performed under ether anesthesia. Consumption of food by tail-cast weight-bearing and suspended animals is similar’ and denervation also did not lower food consumption (Tischler et al, unpublished observation). Animals were killed by cervical dislocation. All procedures used were approved by the University of Arizona Laboratory Animal Care Committee.

Female rats (80 to

757

90 g for in vitro studies

Protein Metabolism Standard procedures for excising and incubating skeletal muscles were used.J.“.” Soleus muscles were excised, weighed, and then placed into 25mL Erlenmeyer flasks fitted with self-sealing stoppers. The flasks contained 3 mL of Krebs-Ringer bicarbonate buffer, pH 7.4, equilibrated with 95% 0,:5% CO?, to which was added 5 mmol/L glucose, insulin as indicated, and 0.5 mmol/L cycloheximide to inhibit protein synthesis. *’ For in vitro protein degradation, muscles were first preincubated for 30 minutes and then incubated in fresh medium for 2 hours. Other agents were added to the medium as indicated in the figure legends. The incubations were terminated by removing the muscle and heating the medium in boiling water for 3 minutes. Release of tyrosine into the medium was taken as a measure of proteolysis.” The in vivo fractional rate of protein degradation (percent/day) was estimated as the difference between the measured fractional rates of synthesis and of the calculated fractional change in protein by content as described below. 4.*8.*9Animals were tranquilized forelimb injection of 0.1 mL/lOO g body weight of 10% (vol/vol) Innovar-vet (4 Kg Sublimaze, 200 rg Inapsine; Pitman-Moore, Washington Crossing, NJ). After 15 minutes the animals were injected intraperitoneally with 300 rmol and 40 ICi of L-sidechain‘H-phenylalanine (ICN Radioisotopes Division, Irvine, CA) administered in 2 mL of 0.9% (wt/vol) saline solution per 100 g body weight. Fifteen minutes later, both soleus muscles were removed within 75 seconds and frozen in liquid nitrogen. Although comparative measurements of the first or second muscle excised show no significant difference in the results,4 we alternated excising the control and experimental muscles when contralateral muscles were treated differently. Specific activities of free intracellular (S,) and protein-bound (S,) phenylalanine were determined as described elsewhere.‘” with modifications restricted to the volumes used.

Lysosomal Latency The aggregate assay of cathepsins B, H. and L was used to evaluate the relative latency of muscle lysosomes under the various conditions essentially as described previously.3’,” After weighing the muscles on a millibalance, a particulate fraction was prepared by first mincing the muscles on ice in 3 mL of 10 mmol/L MOPS, pH 7.4, 20 mmol/L KCl, 1 mmol/L EDTA, 10 mmol/L sodium pyrophosphate, and 250 mmol/L sucrose. The minced muscles were then homogenized on ice in a Dual1 tube using a motor-driven tissue grinder fitted with a ground glass pestle. The homogenate was diluted with ice-cold buffer to give S to 6 mg muscle per milliliter and was then centrifuged at 760 xg for 5 minutes at 4OC. For measuring total cathepsin activities, Triton Xl00 (0.2% final) was added to a portion of the supernatant solution. Then, a 0.5-mL aliquot of the supernatant solution, with or without Triton-treatment, was diluted with 0.5 mL of 50 mmol/L MES, pH 5.5, and 0.5 mmol/L dithiothreitol. The mixture was equilibrated to 37OC and the pH was then adjusted to 5.5 using 2 mol/L HCl(0 to IO rL). To initiate the reaction, a 0.3-mL aliquot of this solution was added to 0.05 mL of 2.1 mg/mL polyGAT (a random polymer of glutamate, alanine and tyrosine: Sigma Chemical, St Louis, MO). After 40 minutes incubation at 37OC. the reaction was terminated by addition of 0.5 mL trichloroacetic acid (40%). Acid soluble tyrosine was assayed in 0.5 mL of the reaction mixture after centrifugation in an Eppendorf tube (5 minutes at 12,000 x g) and in a blank containing the same amount of tissue extract. The tissue extract was assayed for protein by the Lowry procedure. 3o Latency was calculated as the difference in activity in the presence and absence of Triton Xl00 expressed as a percentage of the total activity measured after Triton treatment.” When expressed in this manner a decrease in the value represents a decrease in latency (eg, less activity is “hidden” from the polypeptide substrate).

In Vivo Intramuscular Injections Animals were injected intramuscularly (IM) by a procedure with adapted from Gerard et al. ‘A Rats were lightly anesthetized ether, and after shaving both hindlimbs the skin was swabbed with betadine solution, A 5-mm incision was made in the outside of the limb and a curved forceps used to carefully hook the soleus muscle. Then 2.5 nL of 0.9% NaCl was injected into the left muscle and 2.5 NL of 200 mmol/L mersalyl or chloroquine was injected into the right muscle. The wounds were closed with surgical clips and muscles were excised 24 hours later. The amounts of mersalyl and chloroquine chosen for injection were approximately proportional by muscle size to that injected into gastrocnemius muscles in other studies.“‘,-“’ Initial body mass of weight-bearing animals averaged 5 g smaller, allowing for the muscle masses to be similar at the time of injection. Data for muscle protein content were corrected for this small difference in initial body weight (see in vivo protein metabolism).

TISCHLER

758

Analyses 232 * a

Muscle protein content was assayed by the biuret procedure35 unless otherwise indicated. Tyrosinej’ and phenylalaninez8 were assayed fluorometrically. Testing of significance of differences for multiple groups was done by factorial ANOVA with a post hoc Scheffe F test or by Student’s t test as indicated in each legend. RESULTS

I-

Atrophy caused by denervation is more severe and associated with faster proteolysis than is atrophy induced by unweighting. I6 This was verified by testing the effect of denervation on the unweighted soleus muscle. In a typical experiment, after 6 days the denervated soleus muscle (25 k 1 mg; 4.0 k 0.2 mg protein) was smaller than the contralateral innervated muscle (29 + 1 mg; 4.5 k 0.2 mg protein) of hindlimb-suspended rats.

E

In Vitro Muscle Treatments As hypothesized earlier, lysosomal proteolysis may be accelerated in atrophy induced by denervation but not by unweighting. Consequently, denervated muscle might show faster proteolysis and thus greater atrophy. If this idea is true, then greater in vitro proteolysis in the denervated muscle may be abolished or diminished by lysosomotropic agents. This idea was tested using three agents known to inhibit lysosomal proteolysis: chloroquine, which raises intralysosomal pH and specifically inhibits cathepsin B.37 methylamine, which neutralizes the intralysosomal PH,~* and leucine methylester.39-4’ Proteolysis in the unweighted muscle was 28% to 41% faster (P < .05) than in the weighted muscle, and was even more rapid (P < .05) in the denervated muscle (23% to 31% greater than unweighted) (Fig 1). Chloroquine and leucine methyl ester significantly inhibited protein degradation in control muscles by 16% and 12%, respectively. When the data for the atrophied muscles are expressed as absolute values rather than as percent gain, the following effects become apparent. Chloroquine decreased protein degradation in the unweighted muscle by 12% (from 327 k 23 to 289 + 13 pmol tyrosine/mg muscle/2 hours), and in the denervated muscle by 26% (from 401 * 16 to 295 f 14). Also, in the denervated muscles, methylamine and leucine methylester both significantly inhibited proteolysis by 22% and 28%, respectively. Rates of protein degradation in these muscles were: no addition, 558 + 21; methylamine, 434 + 15; leucine methylester, 400 t 18 pmol tyrosine/mg muscle/2 hours. None of the lysosomotropic agents significantly altered the percent difference in protein degradation between the control and unweighted muscles. In contrast, each agent abolished the difference in proteolysis between the unweighted and denervated muscles. While these results support our hypothesis, an alternate explanation could be that preservation of autophagic vacuoles differed in these muscles during incubation. Thus, the data could reflect a consequence of incubation rather than of in vivo perturbations. Therefore, we measured lysosomal latency of cathepsins B, H. and L (Table 1). Our data for freshly isolated normal muscle (50% k 2%) are similar to that reported by 0dessey3* in the same preparation (43% * 4%). Although incubation tended to diminish lysoso-

60 T

Control

Values

194 AI 4

334 rt_ 9

299 t_ 19

ET AL

294 k 9

r fl -r

N!

NS

Addltlons:

None Expt.

CLQ

I

None

MAM

Expt.

LME

2

Fig 1. Effects of lysosomotropic agents on the response of proteolysis to unweighting (m) or denervation (0) for 3 days. Muscles were incubated in the absence or presence of 0.1 mmol/L chloroquine (CLQ), 10 mmol/L methylamine (MAM), or 10 mmol/L leucine methyl ester (LME). Control values (pmol tyrosine/mg muscle/2 h) are for protein degradation in weight-bearing muscles for each condition. Numbers in the open bars (denervated) give the difference in absolute rate (P < .05) from the values for unweighted muscles. NS means not significantly different from unweighted. In each instance protein degradation was significantly greater W < ,051 in the unweighted or denervated muscles than in the weighted (control) muscles. Data are mean values k SE for muscles from 10 animals.

ma1 latency, the apparent difference was not highly significant (P < .l) and the control value after incubation was still similar to the preincubation latency measured by Odessey. Either before or after incubation (Table 1) lysosomal latency was similar in weighted and unweighted muscles, but was lower in denervated muscles. Since this difference was similar with or without incubation, it is unlikely that artifacts caused by incubation would explain the data in Fig I. The role of the lysosome in accelerated in vitro proteolysis was studied further by comparing the effects of lysosomotropit agents on both lysosomal latency and protein degradation in the same muscles. Under no condition did the total activity of cathepsins B, H, and L in the 760-g supernatant differ between unweighted, denervated, and control muscles (data not shown). Leucine methylester causes osmotic swelling and breakage of the lysosome,40.4’ thus decreasing lysosomal latency (Table l), as reported previously,32 and generally lowering the release of tyrosine from muscle (Fig 1, Table 1). In accord with the greater effect of leucine methylester on proteolysis in denervated than in control or unweighted muscle, this agent tended (P < .l) to lower latency more so in the denervated muscles (Table 1). Since this compound is an indicator of lysosome fragility, the results suggest that lysosomes in denervated muscle may be more fragile and thus were more sensitive to treatment with leucine methylester, but not to incubation without any treatment. To determine whether initiation of autophagy is enhanced in denervated muscle relative to control and unweighted

PROTEOLYSIS IN ATROPHYING MUSCLE

Table 1. Effects of Unweighting

759

or Denervation

for 3 Days and of Leucine Methylester

or Methyladenine

on Lysosomal Latency and

Proteolysis Condltlons

Wwght-bearing

Unwelghted

Denervated

Latency (%I Nonincubated

50 + 2

46 i 3 t-8)

35 k 3t*

Incubated Treatments

39 * 3

34 + 3 (- 13)

27 * 2$ (-31)

No addition

42 k 3

32 ? 2

26 k 3

+Leu-methylester

33 * 2

25 ? 3

17 k 3

Difference (%)

-20

-20

t-301

-35

No addition

37 k 2

35 k 2

28 f 3

+ 3-methyladenine Difference 1%)

46 k 3 f24

42 -r 3

34 k 1

+20

t22

Protein degradation (pm01 tyrosine/mg muscle/2 h) 360 i 20

465

+ Leu-methylester

296 k 18

391 + 25’

Difference (%)

569 k 21tS

+ 19”

No addition

401

?z 22$ -- 30

-16

-18

No addition

335 + 14

451 k 14’

582 + 19t$

+ 3-methyladenine Difference (%)

271 t 13 -19

369 k 18’ -18

448

+ 16t$ -23

NOTE. Latency of lysosmal protease activity (pH 5.5) was measured in freshly isolated or incubated muscle as described in Methods. Muscles were incubated with cycloheximide in the absence or presence of 10 mmol/L leucine methylester (leu-methylester) or 5 mmol/L 3-methyladenine. 3-Methyladenine diminished (f’ < .D5) proteolysis in all muscles, but leu-methylester was only effective in denervated muscles. Numbers in parentheses are percent difference from value in the weighted muscle. Data are mean values +SE for muscles from six to eight animals.

lUnweighted

“weight-bearing by ANOVA.

‘Denervated Y unweighted by ANOVA. ‘Denervated vweighted by ANOVA.

muscle, we treated muscles with 3-methyladenine, a purine which specifically inhibits the first step in autophagy.4’ As reported previously,‘4 3-methyladenine increased lysosomal latency in muscle and coincidentally decreased protein degradation (Table 1). Since this compound had similar effects in all three muscles, it is unlikely that the initiation of autophagy is enhanced by denervation. Because lysosomotropic agents did not attenuate the difference in proteolysis between weighted and unweighted muscles, enhanced protein breakdown with reduced load on the muscle could be caused by accelerated proteolysis in the cytoplasm. Skeletal muscle cytosol contains thiol proteases, especially calpain,4’ whose role in muscle protein degradation remains controversial. To determine whether thiol proteases might be important in the accelerated proteolysis associated with atrophy induced by unweighting, we incubated muscles in the absence or presence of mersalyl (Fig 2). Under such conditions, the calcium-dependent proteolytic activity may be completely inhibited while cathepsin B activity, which is not inhibited in intact muscle, remains unaffected.24,25 As in previous work,24,25 mersalyl did not affect protein degradation in normal muscles. In the absence of mersalyl, protein degradation was faster (+25%) in unweighted than in control muscles and was even greater (+ 60%) in the denervated muscles. Addition of mersalyl abolished the difference in protein degradation between the control and unweighted muscles. Mersalyl only partially inhibited degradation in denervated muscle so that the absolute difference in rate between the unweighted and denervated muscles was maintained. Therefore, thiol proteases seem to be mainly responsible for the accelerated in vitro proteolysis of unweighted muscle.

Control 390 + 20

349 + n

80

Values 378 + Is

390 f 17 T

iL IO'

T

60

1

13

I

I

-I

13(

40

20

1Additions:

-

-

Mersalyl

TMB-8

I J

Fig 2. Effects of mersalyl and TMB-8 on the response of in vitro proteolysis to unweighting (ml or denervation (0) for 3 days. Muscles were incubated in the absence or presence of 0.2 mmol/L merselyl or 0.06 mmol/L TMB8. When TMB-8 was added, CaCI, was omitted from the medium. Control values and numbers in the open bars are as described for Fig 1. Except for mersalyl treatment of unweighted muscles. protein degradation was greater (P < .05) in the unweighted or denervated muscles than in the control muscles. Mersalyl and TMB-8 inhibited (P < ,051 protein degradation in the unweighted and denerveted muscles. Data are mean values + SE for muscles from 10 animals.

TISCHLER ET AL

760

Table 2. Effects of Calcium lonophore A23187

on Protein Degradation

in Weight-Bearing

or Unweighted

Muscles in the Absence or

Presence of Calcium Weight-Bearing ipmol tyrosinelmg muscle/Z h) A23187

Unweighted (pmol tyrosine/mg muscle/Z h)

With Calcium

No Calcium

No Calcium

With Calcwm

None

312 k 14

380 ? 8

390 + 17

456

f

Added

372 c 14

497

502 k 21

641

+ 28

112 k 21 (29)

185 f 29 (41)

Difference NOTE. At 3 days after tail-casting,

60 t

lO(19)

weight-bearing

+ 20

117 + 19 (31) or unweighted

muscles were incubated with 0.5 mmol/L cycloheximide

19

in the absence or presence

of 2 pmol/L calcium ionophore A23 187. When CaCI, was omitted from the buffer, EGTA was provided at 0.5 mmol/L. All effects of A23 187 were paired. Data are mean values *SE for muscles from 10 animals. Data were analyzed by factorial ANOVA. Values given in parentheses are the percent difference. All effects of addition of A23187,

addition of calcium, and unweighting were significant at P < .05.

The role of calcium-activated proteolysis in atrophy caused by unweighting was studied further using two approaches to alter the cytosolic concentration of calcium. In the first experiment, muscles were incubated in the absence of added calcium and with TMB-8, which antagonizes the release of calcium from the sarcoplasmic reticulum.26 TMB-8 did not affect protein degradation in control muscles, but inhibited degradation by 25% in unweighted muscles and by 17% in denervated muscles (Fig 2). Thus, TMB-8 nearly abolished the difference in proteolysis between control and unweighted muscles. but only reduced this difference from 75% to 51% greater in denervated muscles. In the other experiment, calcium ionophore A23 187 was used to increase intracellular calcium. As reported previously,25 the ionophore increased protein degradation whether or not calcium was also provided (Table 2). Presumably this effect in the absence of calcium demonstrates the ability of A23187 to release calcium from intramitochondrial stores44 and/or from the sarcoplasmic reticulum.25 In the absence or presence of calcium, ionophore A23 187 increased protein degradation to a greater extent in the unweighted than in the weighted (control) muscle. Taken together, these experiments suggest that calcium-dependent proteolysis in unweighted muscle may be more sensitive to intracellular changes in calcium than is normal muscle and may play a role in accelerated protein degradation in this muscle.

marked atrophy for this period. Chloroquine treatment did not affect the extent of muscle atrophy between days 2 and 3 of unweighting. However, mersalyl treatment not only abolished this loss of protein, but led to a 5% accretion of protein. In the unweighted muscle, neither chloroquine nor mersalyl affected in vivo protein synthesis, which was consistently lower (32% to 37%) than in the control muscle (Table 3). In unweighted muscles injected with saline or chloroquine, greater protein degradation (54% to 69%) than in weighted muscles accounted for a significant portion of the protein lost by this muscle during this 24-hour period. In contrast, unweighted muscles treated with mersalyl showed slower degradation than control muscles, thereby offsetting the

7

-24

HOURS

n

CLQ-TREATED

m

SALINE

6?i

MERSALYL-TREATED

In Vivo Muscle Treatments Other investigators have successfully injected chloroquine and mersalyl IM to inhibit lysosomal proteolysis’4 and calpain, respectively. Using this approach we tested the in vivo effects of these agents on protein loss and protein metabolism of denervated and unweighted soleus muscles. The periods of study selected, days 2 to 3 after unweighting and days 1 to 2 after denervation, were based on the peak period of acute atrophy identified in other studies (Satarug et al, manuscript submitted).‘(‘Calculations showed that weightbearing muscles injected with saline accumulated about 5.9% protein during the 24 hours (Fig 3, Table 3) compared with 5.3% per 24 hours as we reported for nontreated soleus muscles.4 Injection of chloroquine or mersalyl did not alter this value (Fig 3) or affect in vivo protein synthesis or degradation in the weight-bearing muscle (Table 3). After 2 days of unweighting, the soleus had 8.5% less protein than control (Fig 3). The difference between salinetreated muscles on day 3 increased to 20%, reflecting the

L

WEIGHTED

UNWEIGHTED

DENERVATED

Fig 3. Effects of IM injections of chloroquine or mersalyl on alterations of protein content caused by unweighting or denervation. Muscles were injected as described in Methods and denervated 24 hours before injection. Animals were suspended 48 hours before injection. Animals were weight-matched on day 0. Chloroquine-treated denervated and merselyl-treated unweighted muscles were significantly (P c .05j larger than the contralateral saline-treated muscles by paired f test or by ANOVA. Date are mean values + SE for muscles from eight to 15 animals from two or three experiments.

PROTEOLYSIS IN ATROPHYING MUSCLE

Table 3. Effects of IM Injections

761

of Chloroquine

or Mersalyl on the Responses of In Vivo Protein Turnover

to Unweighting

or

Dernervation. ProteinChanges (%I24 h) SAL

CLQ

ProteinSynthesis (%I24 h) MER

ProteinDegradation 1%/24 h)

SAL

CLQ

MER

SAL

CL0

MER

10.7

10.3

10.1

Weighted

+5.9

f5.9

+7.4

16.6 k 0.8

16.2 2 1.0

17.5 + 0.9

Unweighted

-7.6

-4.9

+5.4

10.5 + 0.9

1 1 .o r 0.4

11.6 5 1.1

Denervated

-3.7

+7.6

-4.6

16.5 + 1.1

15.8 k 0.7

15.7 k 1.8

18.1

15.9

6.2

(+SSl

(+54)

(-33)

8.2

20.3

20.2 (1-89)

t-201

NOTE. Muscles were injected as described in Fig 3. Fractional changes in protein content were calculated as the percent increase

(+118)

(+) or decrease{ -

1

over 24 hours in muscle protein content using the data in Fig 3. A statistical analysis of protein changes IS not possible since growth is based on the mean muscle protein content for each day adjusted for initial animal size.4,s,29In viva protein synthesis was measured in these muscles from eight to 10 antmals at 24 hours after injection. Protein degradation was calculated as the difference between the measured rate of synthesis and the calculated change in protein. Values in parentheses are the percent difference from weighted muscle having the same treatment. Because the protein degradation values are calculated from mean data for synthesis and growth, it is not possible to test these numbers for significance of apparent differences4 Abbreviations: SAL, saline; CLQ, chloroquine; MER, mersalyl.

lower rate of protein synthesis. Thus in vivo, as in vitro, mersalyl could abolish the accelerated proteolysis caused by unweighting. Muscles denervated for 2 days and injected with saline contained about 10% less protein than control muscle at 2 days after tail-casting (Fig 3). In contrast to the results for unweighted muscle, chloroquine abolished this difference in protein content from weighted muscle, while mersalyl did not affect this difference. Chloroquine, like mersalyl, did not affect in vivo protein synthesis (Table 3). Therefore, the ability of chloroquine to prevent protein loss in the denervated muscle was caused by a decline in protein degradation. Proteolysis in denervated muscles injected with saline or mersalyl was much greater than in control muscles. Therefore, these in vivo results with chloroquine treatment of denervated muscle paralleled the in vitro experiments (Fig 1). DISCUSSION

Most investigators compare different models of atrophy by reference to work of other investigators. This study is unique in providing a direct comparison of potential mechanisms of accelerated proteolysis caused by denervation or unweighting of soleus muscle. By comparing mechanisms in these two models, we have gained some insight into the potential and relative roles of lysosomal and cytosolic proteolysis in muscle atrophy. Role of Lysosomal Proteolysis Data herein lend credence to the idea that lysosomal proteolysis is important in atrophy caused by denervation. Thus, lysosomotropic agents markedly diminished protein degradation in denervated soleus muscle in vitro (Fig 1, Table 1). Our ability to prevent further atrophy of the denervated muscle by IM injection of chloroquine suggested that this role of lysosomal proteolysis was not merely an in vitro artifact. Since 3-methyladenine, which inhibits initiation of autophagy, had similar effects on protein degradation and lysosomal latency in all of the muscles (Table l), it is un1ikel.y that this first step in the process was affected by

denervation. Instead the data with leucine methylester suggest that denervated muscle lysosomes may be more fragile and more permeable (Table l), so that proteolysis would be accelerated by making lysosomal proteases more accessible to protein substrates. That lysosomal proteolysis may be relatively unimportant in atrophy caused by unweighting contradicts reports of increased activity of lysosomal proteases in such muscles.6.4s However, measurements of activity expressed only per amount of tissue protein can be misleading. A more suitable approach for considering the change in status of an enzyme is to determine the total activity prior to and after the period of atrophy. Using data from an unweighting study by Goldspink et ah6 we calculated initial and final total activities of cathepsins B and D from the published measurements of specific activity, total tissue protein, and the normal muscle rate of growth. According to this approach, unweighting atrophy seemed to cause a loss of total activity of both cathepsins B and D. A similar calculation from data for lysosomal (acid) protease activity,46 indicated no change in total activity at 4 days of unweighting with a lower total activity on day 14. These data do not support an increased role of lysosomal proteases in unweighted muscles. In contrast, similar calculations of total cathepsin D activity in denervated muscle indicated that the loss in mass could not account for the increase in specific activity? Role of Cytosolic Proteolysis The data in this study suggested that cytosolic proteolysis is important in unweighting atrophy. This idea was supported by the ability of mersalyl to abolish the difference in proteolysis both in vitro (Fig 2) and in vivo (Table 3) between the weighted and unweighted muscles. Mersalyl does not inhibit lysosomal proteases but inhibits calcium-activated protease (calpain) in incubated muscle.24,‘5 Therefore, its effect is mediated by inhibition of thiol protease in the cytosol, possibly including calpain. These ideas agree with observations that lysosomal autophagy was not evident and that the activity of calpain increased following hypogravityinduced atrophy of the soleus muscle.47 Further evidence for

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a role of calcium-dependent proteolysis and possibly for calpain were obtained using calcium ionophore A23187, which produced a greater effect on protein degradation in the unweighted than in the control muscle (Table 2). Furthermore, a role of calcium stored in the sarcoplasmic reticulum was supported by TMB-8 diminishing accelerated protein degradation in the unweighted muscle (Fig 2). The specific role of calcium-dependent proteolysis in muscle atrophy remains controversial. Following denervation, calcium increases in conjunction with cellular necrosis,48 but the mechanism by which this calcium stimulates protein degradation is unclear. Some studies of this mechanism support a role for nonlysosomal proteases,49-5’ in particular cytosolic thiol proteases.50.5’ Although studies with mersalyl suggested that calcium-activated protease was not the likely target for this effect of calcium,” Zeman et al49 proposed that a sarcoplasmic low (micromolar) calcium-activated protease might be inaccessible to mersalyl, and therefore still a possible mediator of calcium-dependent proteolysis. Provid-

ing further confusion are recent reports that elevated intracellular calcium is responsible for the increased breakdown of nonmyofibrillar but not myofibrillar proteins.5’,53 Therefore, the significance of elevated calcium in necrosis associated with denervated muscles4* remains uncertain. CONCLUSION

These results can form the basis for addressing important issues regarding the physiological adaptations to and biochemical mechanisms of muscle atrophy caused by unweighting or denervation. The data support the hypothesis that there are major fundamental differences in these mechanisms. There is a strong possibility of a role for lysosomal proteolysis in atrophy induced by denervation but not by unweighting. Therefore, a significant problem to be pursued is a novel role for innervation in attenuating lysosomal proteolysis. Further, it is of interest to investigate in these muscles the potential roles of calcium-dependent and thiol protease-mediated proteolysis.

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Different mechanisms of increased proteolysis in atrophy induced by denervation or unweighting of rat soleus muscle.

Mechanisms of accelerated proteolysis were compared in denervated and unweighted (by tail-cast suspension) soleus muscles. In vitro and in vivo proteo...
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