proteolytic degradation of isolated myofibrils and myofibrillar proteins by m-calpain from the...

THE JOURNAL OF EXPERIMENTAL ZOOLOGY 27630-42 (1996)

Proteolytic Degradation of Isolated Myofibrils and Myofibrillar Proteins by m-Calpain From the Skeletal Muscle of the Amphibian Rana ridibunda

rq. SARGIANOS, c. GAITANAKI, B. DIMITRIADIS, AND I. BEIS Laboratory of Animal Physiology, Department of Zoology (N.S., C. G., I.B.) and Laboratory of Biology, Department of Genetics, Developmental and Molecular Biology (B.D.), School of Sciences, Aristotle University of Thessaloniki, Thessaloniki 54006, Greece

ABSTRACT The effects of m-calpain isolated from the skeletal muscle of Rana ridibunda on the myofibril and inyofibrillar proteins were examined. The results of this study clearly showed that treatment of 1myofibrils with calpain in the presence of 2 mM Ca2+ results in the complete disappearance of Z-lines within 5 min, whereas prolonged incubation results in the appearance of single sarcomeres or groups of 2-5 sarcomeres. From the other divalent cations examined only Srz' at high concentration induces the Z-line removal by calpain. Presence of Mn2+ or Ba2+ reduces the Ca2+ requirement of calpain to the Z-line removal. Protease inhibitors such as endogenous calpastatin, E-64, and leupeptin completely inhibit the Z-line removal by calpain, whereas PMSF and trypsin inhibitor do not inhibit the proteolytic activity of calpain. Studies on the proteolytic degradation of various myofibrillar proteins isolated from the skeletal muscle of Rana ridibunda showed that myosin and G-actin could represent "good" substrates of calpain, whereas F-actin and tropom,yosin are not degraded by this protease. Our results also showed that the optimum conditions of calpain action and function on the myofibrillar protein degradation are quite similar to those described for both its maximal caseinolytic activity (Sargianos et al. [1994] J. Exp. Zool., 269:95-105) and autolytic modification (Sargianos et al. [19951 J. Exp. Zool., 271:82-94). 0 1996 Wiley-Liss, Inc.

Since the first description of a calcium-activated cysteine protease (calpain; CANP [EC 3.4.22.171) in rat brain (Guroff, '64) calpain activity has been identified in most if not all tissues and cell types of vertebrates (Murachi et al., '81; Murachi, '83; Pontremoli and Melloni, '86; Croall and DeMartino, '91). Calpain now forms a family consisting of at least six distinct members (Sorimachi et al., '139, '93; Saido et al., '94). Detailed studies on the mammalian calpains have shown that the calpain members can be divided into two groups, ubiquitous and tissue specific calpains (Sorimachi et al., '94; Saido et al., '94). Studies on the purified ubiquitous p- and m-calpains from various sources have shown that these calpains differ in their sensitivity to Ca2+; p-calpain requires low Ca2+ whereas m- calpain requires high (;a2+ for both their half maximal and maximal activities, respectively. Both enzymes are heterodimers, each consisting of a large catalytic (approximately 80 kDa) and a small regulatory (approximately 30 kDa) subunit. Both p-calpain and m-calpain seem to

0 1996 WILEY-LISS, INC.

undergo autolysis in the presence of Ca", producing enzymes with higher sensitivity to Ca2+ than the unautolyzed enzymes (Dayton, '82; Hathaway et al., '82; Coolican and Hathaway, '84; Inomata et al., '85; Suzuki et al, '88; Ed- munds et al., '91; Nishimura and Goll, '91).

In spite of the detailed studies on the molecular and regulatory characteristics of mammalian and avian calpains in vitro, the real physiological functions of the calpain-calpastatin system remain obscure.

In the skeletal muscle, they appear to play an important role in nonlysosomal, specific, and limited degradation of various myofibrillar proteins (Ishiura, '81; Barrett et al., '91). Previous studies by several investigators had demonstrated the incubation of isolated myofibrils with calpain in the presence of Ca2+ resulted specifi-

Received July 13, 1995; revision accepted April 3, 1996. I. Beis is presently at Laboratory of Animal Physiology, De-

partment of Zoology, School of Sciences, University of Ath- ens, Panepistimioupolis 15784, Athens, Greece. Address reprint requests there.

MYOFIBRIL DEGRADATION BY M-CALPAIN 31

cally in the removal of Z-lines (Busch et al., '72; Reddy et al., '75; Dayton et al., '76a,b). Subse- quent studies had reported that many pathological conditions characterized by increased protein catabolism also appear to be associated with increased intracellular calcium levels and increased calpain activity. For example, increased calpain activity was measured in atrophying muscle caused by vitamin E deficiency (Dayton et al., '79), in Duchenne muscular dystrophy (Kar and Pearson, '76), and various cardiomyopathies (Rudge and Duncan '84). Although calpain seems to act very specifically to the limited disruption or complete disappearance of Z-lines in damaged or hypertrophic skeletal or cardiac muscle, Z- lines also may be drastically altered under physiological conditions and it seems possible that the calpain may participate in myofibrillar assembly or disassembly. The newly discovered n-calpain 1 is expressed exclusively in the skeletal muscle, suggesting its muscle-specific roles (Sorimachi e t al., '89). The involvement of calpain in various pathological states such as muscular dystrophy (Turner et al., '88) and ar- thritis (Yamamoto et al., '92) is of particular clinical interest and further research may lead to therapeutic applications.

Dayton et al. ('76a,b), Ishiura et al. ('791, and Sugita et al. ('80) have demonstrated that purified calpain digested troponin, tropomyosin, C-protein, and myosin heavy chain by limited proteolysis. Many investigators have reconfirmed these results in cardiac (Toyo-oka et al., '78; Toyo-oka and Masaki, '79) and skeletal muscle (Azanza et al., '79, SO).

Studies on the immunocytochemical localization of calpain have shown that calpain is located in or near the Z-line (Ishiura et al., '80; Dayton and Schollmeyer, '81; Yoshimura et al., '86), a myofibrillar structure very susceptible to degradation by this protease. Localization studies have also shown that calpain is present in the cytoplasm and in or near the plasma membrane in both muscle and non-muscle cells (Barth and Eke, '81; Dayton and Schollmeyer, '81) suggesting that calpain may also be involved in the degradation of cytoplasmic, cytoskeleton, and membrane proteins.

In our previous papers in this journal, we had described the purification and characterization of m-calpain from the skeletal muscle of the amphibian Rana ridibunda, as well as our studies on the autolytic mechanisms responsible for the regulation of this purified calpain in vitro

(Sargianos et al., '94, '95). The results of these studies showed that Rana ridibunda skeletal muscle calpain activity may be regulated by both autolytic mechanisms and synergistic effects of various divalent cations such as Ca2+/Mn2+ or/ and Ca2+/Sr2+. In the present study we describe the possible physiological function of calpain- calpastatin system in the myofibrillar degradation in the skeletal muscle of Rana ridibunda.

MATERIALS AND METHODS Materials

Unless indicated, all chemicals and reagents were purchased from Serva (Heidelberg, Ger- many) or Sigma Chemical Co. (St. Louis, MO) in the highest grade available. Casein (Ham- marsten grade) was obtained form Merck (Darm- stadt, Germany).

Preparation of calpain m-Calpain was purified from -600 g of skeletal

muscle of the amphibian Rana ridibunda as previously described (Sargianos et al., '94) and stored in 20 mM Imidazole/HCl buffer, pH 7.2, which contained 1 mM EDTA, 1 mM EGTA, 10 mM 2-mercaptoethanol, and 50% (v/v) glycerol at 4°C. One unit of calpain was defined as a change of 1.0 at 750 n d 3 0 min at 25"C, using alkali dena- tured casein as substrate (Sargianos et al., '94). The specific activity of the purified m-calpain was 316.6 U/mg of protein.

Preparation of calpastatin Calpastatin was partially purified from the skel-

etal muscle of the amphibian R. ridibunda according to the method described by Murachi et al. ('81). The calpastatin fraction recovered from the first DEAE Sepharose CL-6B ion exchange chromatog- raphy was heated at 95°C for 20 min and centri- fuged at Z0,OOOg for 20 min. The supernatant was collected and dialyzed overnight against an ex- cess of 50 mM Imidazole/HCl buffer, pH 7.2, which contained 10 mM 2-mercaptoethanol. The dialy- sate was then concentrated by ultrafiltration using an Amicon (Danvers, MA) membrane (PM 30) and stored at -20°C in 50% (v/v) glycerol. One unit of calpastatin was defined as the amount of protein that inhibits 1 U of calpain under standard assay conditions (Sargianos et al., '94).

Preparation of myofibrils Myofibrils were isolated from the skeletal

muscle of R. ridibunda according to the method

32 N. SARGIANOS ET AL.

described by Ferenczi et al. ('78) with slight modifications. Animals were anaesthetised by im- mersion in 0.01% (w/v) MS-222 for 10 min, and the back and leg muscles were removed and kept on ice. Muscles were lblended in 3 volumes of ice-cold 5 mM Na2HP0,JNaH2P04 buffer, pH 7.0, which contained 40 mM NaC1, 1 mM MgC12, 0.1 mM DTT, and 0.1 mM EDTA, and homogenized in a Sorval Omnimixer using three 30 sec bursts at medium speed. After centrifugation in a Sorvall RC5C centrifuge (Rotor type SS-34), at 6,OOOg for 10 min, the supernatant was discarded and the myofibrils were suspended in 6 volumes of the same buffer. This operation was repeated 5-6 times, in order to remove various cytosolic proteins, nucleic acids, and other substances, and the final myofibrillar pellet was resuspended in 10 volumes of 50 mM Imidazole/HCl buffer, pH 7.2, which contained 10 mM 2-mercaptoethanol.

Preparation of myofibrillar proteins Myosin was prepared from the isolated myo-

fibrils according to the method described by Ferenczi et al. ('78). F-actin, G-actin, and tropomyosin were prepared according to the method of Spudich and Watt ('7 1).

Effect of calpain on the isolated myofibrils Myofibrils were assayed under the following

conditions: 10 mg/ml myofibrils, 100 mM KC1, 50 mM Imidazole/HCl buffer, pH 7.2, 2 mM CaC12, 10 mM 2-mercaptoethanol in a total volume of 0.5 ml. The enzyme-to-substrate ratio was kept at 1500 (w/w). The reaction was started by the addition of 10 pg of purified calpain. The incubation was performed for 30 min at 25"C, and the reaction was terminated by the addition of enough concentrated EDTA to bring the final EDTA concentration to 10 mM. As controls, myofibrils incubated (I) in the presence of 10 mM EDTA instead of CaC12, (2) in the presence of 2 mM CaC12 without calpain, and (3) in the presence of 10 mM EDTA without calpain were included. Then the suspension was diluted with 1 ml of 100 mM KC1 and subjected to a brief homogenization. The myofibrils were then examined with a phase-contrast light microscope to de- termine the effects on rjtructure and degree of f r agmen ta t ion .

Electron microscopy The myofibrils were fixed in Karnovsky's fixa-

tive (Karnovsky, '65) post-fixed in 2% (v/v) os- mium tetroxide, dehydrated, and embedded in

Spurr's resin. Sections were cut using Reichert OmU3 ultramicrotome, post-stained with ura- nyl acetate and lead citrate, and examined under a JEOL lOOB electron microscope operating at 80 KV. For light microscopic observations, thick sections were stained with 1% (v/v) Tolui- dine blue.

SDS polyacrylamide gel electrophoresis Samples were mixed with an equal volume of

a sample buffer which contained 125 mM Tris/ HCl bufTer, pH 6.8, 20% (v/v) glycerol, 10% (v/v) 2-mercaptoethanol, 4% (w/v) SDS, 10 mM EDTA, and 0.02% (w/v) bromophenol blue, and the mixtures were boiled for 2 min before electrophoresis. Polyacrylamide slab gel electrophoresis in the presence of 0.1% (w/v) SDS was performed according to the method described by Laemmli ('70) with 7.5% or 9% separating and 5% stacking gels. Gels were stained for 6-8 hours in 0.1% (w/ v) Coomassie Brilliant blue R-250, 50% (v/v) methanol-10% (v/v) acetic acid solution, and were then destained with 50% (v/v) methanol- 10% (v/v) acetic acid solution. The molecular weight markers used were: P-amylase (200 kDa), phosphorylase a (98 kDa), fructose-6- phosphate kinase (84 kDa), bovine serum albumin (66 kDa), pyruvate kinase (58 kDa), carbonic anhydrase (29 kDa), and soybean trypsin inhibitor (21 kDa).

Protein determination The concentration of protein was determined

by the method of Lowry et al. ('51) using bovine serum albumin as the standard.

RESULTS Effects of calpain on intact myofibrils

Phase contrast microscopy Calpain was examined systematically with re-

spect to its effectiveness in altering the structural integrity of the myofibrils under various conditions. In Figure 1 the time course of m- calpain effect on the isolated myofibrils is shown. As can be seen, myofibril fragments without incubation with calpain, were fairly long and contained intact Z-lines (Fig. la). Treament of myofibrils with calpain for 5 min at 25"C, in the presence of 2 mM Ca" resulted in the disappearance of Z-lines with little other ultrastructur- ally detectable change (Fig. lb). As the incubation time with calpain increased up to 15 min, myofibrils apparently showed another

a

b

MYOFIBRIL DEGRADATION BY M-CALPAIN

Omin

5 min

15min C

d

e

30min

Control

33

Fig. 1. Phase micrographs of isolated myofibrils treated with purified calpain and brief homogenization. Myofibrils were incubated with calpain (500:l wt/wt) in the presence of 100 mM KCl, 50 mM Imidazole/HCl buffer, pH 7.2, lO mM 2-mercaptoethanol, and 2 mM Ca2' for increasing times; (a) myofibrils without any preincubation; (b) after incubation for 5

min, (c) after incubation for 15 min; (d) after incubation for 30 min; (e) after incubation in the presence of 2 mM Ca2+ without calpain, for 30 min. Arrows represent positions of Z- lines or positions from which Z-lines were removed. Magnifi- cation, ~1 ,200 .

type of situation; sarcomeres were shorter, Z- lines had completely disappeared, and empty spaces produced between the sarcomeres (Fig. lc). Prolonged treatment of myofibrils with calpain for 30 min resulted in the appearance of single sarcomeres or groups consisted of 2-5 sarcomeres (Fig. Id). As controls, myofibrils incubated in the presence of 2 mM Ca2' without calpain, for 30 min, were used (Fig. le).

In Figure 2 the effect of m-calpain on the isolated myofibrils in the presence of various divalent cations is shown. As can be seen, Ca2' at low concentration (200 pM) (Fig. 2a) was ineffective, while high concentration (2 mM) of Ca2' induced myofibril fragmentation and disappearance of Z-lines (Fig. 2b). From the other divalent cations tested, only Sr2+ at a concentation of 10 mM was effective (Fig. 2c), whereas Ba2+

or Mn2' even at high concentrations (10 mM for Ba" or 1 mM for Mn2+, respectively) were ineffective (Fig. 2d for Ba", and Fig. 2e for Mn2+, respectively). Incubation of myofibrils with calpain in the presence of 200 KM Ca2' plus 1 mM Mn2+ (Fig. 2 0 or 200 pM Ca" plus 10 mM Ba" (Fig. 2g) at concentrations at which these divalent cations did not show any effect, resulted in the complete disappearance of Z-lines, indicating the synergistic effect of these divalent cations among Ca2+.

We also examined if the apparent fragmentation of myofibrils by calpain is inhibited by various protease inhibitors. The results of this study are shown in Figure 3. As can be seen, 20 pg of partially purified calpastatin (Fig. 3a), or high concentration of E-64 or leupeptin (Fig. 3c, for E-64, and Fig. 3d, for leupeptin, respectively) completely


200pM Ca2+ a 2mMCa2*

b

C

e

lOmM Sr2+ 1OmM Ba2* d

1mM Mn2+ 200pMCa2++ 1mM Mn2+ f

200pM Ca2++10mMBa2+ g

Fig. 2. Phase micrographs of myofibrils treated with calpain (500:l wt/wt) in the presence of various divalent cations. The incubation was performed at 25°C for 30 min in the presence of: (a) 200 pM Ca2+; (b) 2 mM Ca2+; (c) 10 mM

Sr2+; (d) 10 mM Ba+'; (e) 1 mM Mn2+; (0 200 Ca2' plus 1 mM Mn2+; (g) 200 pM Ca2+ plus 10 mM Ba2+. Magnification, ~1,200.

inhibited the fragmentation of myofibrils and Z- line removal by calpain. On the contrary, treatment of myofibrils with calpain in the presence of low concentration (1 pM) E-64 (Fig. 3b), or high concentration (2 mM) PMSF (Fig. 3e) resulted in the complete disappearance of Z-lines. As controls, myofibrils treated with calpain in the presence of 10 mM EDTA instead of Ca2+, or treated with Ca2' in the absence of inhibitors were used (Figs. 3f and 3g, respectively). The above results indicate that the observed fragmentation of myofibrils and complete diisappearance of Z-lines by calpain is specifically inhibited only by cysteine protease inhibitors.

Electron microscopy The ultrastructural examination revealed that

among all myofibrillar structures, the Z-line was the first one which displayed degradative changes. Electron micrographs of longitudinally sectioned

myofibrils incubated with purified calpain in the presence of 2 mM Ca2+ for 15 min revealed that the Z-lines were partially removed (Fig. 4b) com- pared t o the controls (Fig. 4a). In addition, calpain degradation for 15 min produced empty spaces between the sarcomeres, while thin filaments were still present in the I band and between thick filaments. Prolonged incubation of myofibrils with calpain for 30 min, in the presence of 2 mM Ca2+, resulted in the complete removal of Z-lines (Fig. 4c). In the latter case, thin filaments partially disappeared in both the I band and between thick filaments, indicating that calpain effect is a time dependent process. Similar results with those induced by calpain in the presence of Ca2+ were noticed in the presence of 10 mM Sr2+ (Fig. 5a), whereas other divalent cations such as Mn2+, or Ba2+ did not affect the general myofibrillar structure (data not shown). Experiments on the effect of various pro-


C alpastat in( 2 0 ~ 9 ) PMSF( 2 mM 1 a e

E-64( 1 MM 1 b Control

f

C

Control 4

Leupeptin (100pM 1 d

Fig. 3. Phase micrographs of myofibrils treated with calpain (5003 d w t ) in the presence of various protease inhibitors. The incubation was performed at 25°C for 30 min in the presence of: (a) 20 pg calpastatin; (b) 1 FM E-64; (c) 100 yM E-64; (d) 100 yM leupeptin; (e) 2 mM PMSF. As controls,

myofibrils incubated with calpain in the presence of 10 mM EDTAinstead of Ca2' (0 or myofibrils incubated with calpain in the absence of any inhibitor (8) were used. Magnification, ~1,200.

tease inhibitors showed that trypsin inhibitor (200 pg/ml) and PMSF (2 mM) did not inhibit the induced by calpain removal of Z-lines (Fig. 5b), whereas, E-64 at high concentration completely inhibits calpain degratative effect (data not shown), indicating that this effect is not a general proteolytic process but rather a specific, limited degradation of specific myofibrillar structures located in the 2-lines.

Effect of calpain on the isolated myofibrillar proteins

Various myofibrillar proteins such as myosin, G-actin, F-actin, and tropomyosin were isolated from the skeletal muscle of the amphibian R. ridibunda and used as endogenous substrates of

calpain. Our studies on myosin degradation by calpain showed that myosin is degraded in all temperatures examined (0, 15, and 25°C) with a temperature dependent rate (data not shown). In addition, myosin was degraded in all ratios of protease to substrate tested, from 1 : l O up to 1:200 (wt/wt) (data not shown). When the Ca2' requirement inducing degradation of myosin by calpain was examined, it was found that up to 200 pM Ca2' calpain was ineffective, whereas at higher Ca2+ concentrations, calpain induced myosin degradation, reconfirming that the enzyme belongs to the m-calpain subclass (Fig. 6). The electrophoretic pattern obtained clearly shows that myosin heavy chain is degraded producing three groups of polypeptides of lower molecular


Fig. 4. Electron micrographs of isolated myofibrils sectioned longitudinally; (a) untretaed myofibrils; (b) myofibrils treated with m-calpain in the presence of 2 mM Ca2+ for 15 min display partial digestion of Z-line filaments (arrows), while I bands contain thin filament?: between thick ones; (c) myo-

fibrils incubated with m-calpain in the presence of 2 mM Ca2' for 30 min show single sarcomeres isolated by calpain digestion, as well as degradation of many thin filaments in the I band (arrrows). Magnifications: (a) ~17,500; (b) ~20,000; (c) ~25,000.

weights (Fig. 6). Various protease inhibitors were also examined for their ability to inhibit myosin degradation by calpain. The results of this study showed that E-64, leupeptin, and antipain at low concentration (I1 yM) had no inhibitory effect, whereas at higher concentration (1 mM) they completely inhibited this degradation (Fig. 7A, lanes 5-8, for E-64 and leupeptin, and Fig. 7B, lanes 5-6, for antipain, respectively). On the contrary, PMSF (2 mhl), trypsin inhibitor, and chymotrypsin inhibitor (100 or 200 pg/ml) even at high concentrations did not show any inhibi-

tory effect, indicating that myosin degradation is a specific, calpain mediated process (Fig. 7A, lanes 9-10 for PMSF, and Fig. 7B, lanes 7-10, for trypsin and chymotrypsin inhibitors, respectively).

In Figure 8 the effect of calpain on the degradation of myosin isolated from Rana ridihunda skeletal muscle (Fig. 8A) or cardiac muscle (Fig. 8B) is shown, The elctrophoretie pattern obtained clearly shows that myosin heavy chain from these tissues is degraded in a different man- ner. Skeletal muscle myosin is degraded by


Fig. 5. a: Longitudinally sectioned myofibrils isolated from the skeletal muscle of R. ridibundu, incubated with m-calpain in the presence of 10 mM Sr2+ for 30 min, display structural disruption of Z-line filaments (arrows), while thin filaments

are still present in I bands; b: myofibrils incubated with m- calpain in the presence of 200 Fg/ml trypsin inhibitor show complete digestion of Z-line filaments, while the I bands are unchanged. Magnifications: (a) ~25,000; (b) ~20,000.

calpain producing three groups of polypeptides, whereas cardiac muscle myosin degradation produces only one group of polypeptides. These results indicate that the degradation of the endogenous substrate myosin is not a general proteolytic but rather a tissue specific process.

Studies on the tropomyosin degradation by calpain showed that this myofibrillar protein is not degraded by calpain even at a protease to substrate ratio of 1 : l O (Fig. 8C). Experiments on the effect of calpain in the F-actin degradation revealed that this substrate is not degraded by calpain, indicating that it does not represent another endogenous substrate of calpain (Fig. 8D).

On the other hand, our studies on the G-actin degradation by calpain, showed that this endogenous substrate is degraded by calpain even at a protease to substrate ratio of 1:ZOO (Fig. 9). Our previous experiments on the time course of G-actin degradation by calpain had shown that the degradation of G-actin occurs in parallel with

the limited autolysis of the protease and is com- pleted within 20 min (Sargianos et al., '95).

DISCUSSION The removal of Z-lines of myofibrils by calpain

is the most characteristic example of calpain function in both mammalian and avian skeletal muscle. This function has been reported by many investigators (Busch et al., '72; Reddy et al., '75; Dayton et al., '76a,b, '81; Muguruma et al., '80; Hattori and Takahashi, '82; Reddy et al., 83; Zeece et al., '86; Go11 et al., '91). Concomitantly with the loss of Z-lines, a-actinin is removed (Reddy et al., '75; Dayton et al., '76a,b; Zeece et al., '86; Go11 et al., '91).

Although the Ca2+-induced weakening of Z-lines is often confused with the removal of Z-lines by calpain, it has been demonstrated that these processes are clearly distinguishable by the use of muscle calpastatin and various cysteine protease inhibitors such as E-64, leupeptin, and iodoacetate (Hattori and Takahashi, '82; Reddy et al, '83).

38

200 kDa b

78 kDa b

N. SARGIANOS ET A L

1 2 3 4 5 6 7 8 Q 1 0 1 2 3 4 5 6 7 8 9 1 0

m k h b

0.2 0.3 0.4 0.5 0.6 0.8 1 2 1

h 2 + ( m M )

Fig. 6. Changes of the electrophoretic pattern of myosin during degradation by calpain in the presence of Ca2+. Puri- fied myosin (30 kg) was incubated with calpain (0.3 pg) in the assay mixture which contained 50 mM ImidazoldHCl buffer, pH 7.2, 100 mM KCl, :LO mM 2-mercaptoethanol, and increasing concentrations (0.2-2 mMj of CaC12, in a total volume of 30 ~ 1 , at 25°C for 30 min. The reaction was terminated by addition of an equrtl volume of sample buffer for SDS-PAGE. Then the samples were boiled for 2 min, and elec- trophoresed on a 9% polyacrylamide gel, as described in Ma- terials and Methods. Lanes: 1, intact calpain; 2, myosin incubated with calpain in the absence of CaC12; 3-10, myosin incubated with calpain in the presence of increasing concentrations of CaC12, respectively.

Other investigators have studied the effects of temperature and pH on the removal of Z-lines by calpain and showed that the protease retains its activity at low temperatures and pH 6.5 (Zeece et al., '86). Furthermore, Muguruma e t al. ('80) showed that incubation of intact myofibrils with 1 mM Mg2+-ATP induced their contraction, with the characteristic contr(action bands, while incubation of myofibrils with calpain resulted in the removal of Z-lines, a-actinin, and several other proteins preventing the formation of contraction bands.

Our studies on the fragmentation and disruption of Z-lines of the isolated myofibrils from the skeletal muscle of the amphibian R. ridibunda revealed that the optimum conditions of calpain action and function are quite similar to those described for its maximal caseinolytic activity (Sargianos et al., '94).

The results of our studies showed that the removal of Z-lines from in tact myofibrils by calpain in the presence of Ca2+ is a time dependent process, it depends on the Ca2+ concentration, is activated only by Ca2+ rind unphysiological Sr2+

A

,Qool 1 ,poor 1 , , l 2 , € 4 4 Lwpaptin PMSF

(mM) B

,o.m 1 ,*loo 200, !oo 200 , Antipain trypsin chymotrypsin

(mM) inhibitor inhibitor

( P S l m l )

Fig. 7. Changes of the electrophoretic pattern of myosin during degradation by calpain in the presence of various protease inhibitors A Effect of E-64, leupeptin, and PMSF. Lanes: 1, intact calpain; 2, intact myosin; 3, myosin (30 pgj plus calpain (0.3 pg) without any preincubation; 4, myosin (30 pg) plus calpain (0.3 pg) incubated at 25°C for 30 min in the absence of any inhibitor; 5 4 , effect of E-64; 7-8, effect of leupeptin; 9-10, effect of PMSF. B: Effect of antipain, trypsin inhibitor, and chymotrypsin inhibitor. Lanes: 1, intact calpain; 2, intact myosin; 3, myosin (30 pg) plus calpain (0.3 pg) without any preincubation; 4, myosin (30 pgj plus calpain (0.3 pg) incubated at 25°C for 30 min in the absence of any inhibitor; 5-6, effect of antipain; 7-8, effect of trypsin inhibitor; 9-10, effect of chymotrypsin inhibitor.

concentration and by the synergistic effect of Ca2+/Mn2+ or Ca2+/Ba2+; calpain effect is strongly inhibited by partially purified calpastatin from the same tissue and various cystein protease inhibitors. We should notice that prolonged incubation of myofibrils with calpain results not only in the complete removal of Z-lines, but also in the appearance of single sarcomeres or groups of 2-5 sarcomeres (Figs. 1, 4). Our results are in


( A ) Skeletal muscle Cardiac muscle IBI

1 2 %MYOSIN '

1 2

4 2OOkDa b

4 78kDa b

(C 1 TROPOMYOSIN 1 2

4 78kDa c

1 2 3 4 5 6 7 8 9 1 0

78kDa b

45 kDa b

Fig. 9. Changes of the electrophoretic pattern of G-actin after incubation with calpain. Enzymehubstrate ratios (wff wt) tested were: 1:200, 1:150, 1:100, 1:50, 1:25, 1:20, and 1 : l O (lanes 4-10, respectively). Lane 1, intact calpain; lane 2, intact G-actin; lane 3, G-actin incubated with calpain in the absence of CaC12. F-ACTIN ( 0 )

1 2

45kDa b

4 32kDa

Fig. 8. Changes of the electrophoretic pattern of vs.rious myofibrilllar proteins after their incubation with calptiin in the presence of Ca2+. Each isolated myofibrillar protein (30 kg) was incubated with calpain (3 kg) in the standard assay mixture, at 25°C for 0 min (lane 1) or 30 min (lane 50, respectively, and subjected to SDS-PAGE as described i n the text. Effect of calpain on myosin isolated from R. ridibunda skeletal muscle (A) or cardiac muscle (B), and on tropcimyo- sin (C) and F-actin (D) isolated from R. ridibundu skeletal muscle.

accordance with those reported for the effecs of m-calpain on the mammalian myofibrils (Reddy et al., '83; Zeece et al., '86; Go11 et al., '91).

Several studies on the effects of calpain in the degradation of myofibrillar proteins have shown that troponin-T, troponin, I, a-connectin, and nebulin represent "good endogenous substrates of this protease. It has been also reported that calpains are the only proteases which do not. degrade myosin (Dayton et al., '81; Hara et al., '83; Croall and DeMartino, '84; Wang et al., '89; Wolfe et al, '89; Barrett et al., '91; Go11 et al., '9 l), although some investigators suggest i;hat

myosin heavy chain is degraded by this protease (Dayton et al., '76a; Ishiura et al., '79; Sugita et al., '80, Reddy et al., '83).

In the present study we examined in detail the effect of R. ridibunda skeletal muscle calpain on the degradation of the isolated myosin from the skeletal and cardiac muscle of R. ridibunda. The results of these studies revealed that: (1) myosin is degraded by calpain in all enzyme to substrate ratios tested (from 1 : l O up to 1:200 by weight) and that the degradation rate depends on the enzyme t o substrate ratio (data not shown); (2) myosin degradation occurs in all temperatures tested (0, 15, and 25"C), with a temperature dependent rate (data not shown); (3) the Ca2' requirement of calpain for the degradation of myosin is higher than 200 pM (Fig. 6); (4) known cystein protease inhibitors such as E- 64, leupeptin, and antipain completely inhibit myosin degradation by calpain, whereas other protease inhibitors such as trypsin and chymotrypsin inhibitors, and PMSF exhibit no inhibitory effect on the proteolytic activity of calpain (Fig. 7); and (5) comparative studies of myosin degradation from the skeletal and cardiac muscles d R. ridibunda by calpain shows the different susceptibility of these myosin types derived from the respective tissues for proteolysis by the same protease (Fig. 8A, and B, for the skeletal muscle, and cardiac muscle myosin, respectively).

The fact that there was less or no degradative effect on the thick myofilaments might be ex- plained by the difference in the conformation of myosin in situ and in the purified form. Although


there are opposite reports for the degradation of myosin by calpain, Hara et al. ('83) showed the different electrophoretic profiles of myosin degradation polypeptides from the skeletal and cardiac muscles of monkey by the skeletal muscle calpain from the same species. Furthermore, Reddy et al. ('831, by the use of a sensitive electrophoretic method, have shown that myosin from rabbit muscle alslo is degraded by calpain, while there is no apparent effect of the protease on the thick myofilament ultrastructure.

Our studies on the dlegradation of the isolated F- and G-actin from t'he skeletal muscle of R. ridibunda by calpain revealed that although G- actin is fully degraded (Fig. 9), F-actin is not susceptible to degradation by this protease, even at low enzyme to subslsate ratio ( 1 : l O ) after incubation for 30 min (Fig. 8D). These findings seem to be inconsistent with our finding that I band of intact myofibrils seems to be disoriented after incubation with calpain in situ. However, re- cent studies by Go11 e t al. ('91) have shown that incubation of calpain with mixtures of purified a-actinin and F-acttin has no effect on either a-actinin or actin, whereas it specifically removes the Z-lines from intact myofibrils of rabbit skeletal muscle, as well as the a-actinin in situ. The nature of this difference is unclear, but it may be suggested that addiiional proteins susceptible to degradation by calpain may extend into the Z-lines of skeletal muscle myofibrils, such as eu- actinin (Kuroda et al., '811, zeugmatin (Taka- hashi and Hattori, '8!3), and nebulin (Nave et al., '90). On the otheir hand, our finding that calpain fully degrades the isolated G-actin is in accordance with previously reported studies on the rabbit skeletal muscle G-actin (Reddy et al., '83) and it seems likely that after the removal cf a-actinin from the Z-line, which is necessary for the elongation/polymerization of G-actin, unpolymerized G-actjn could be degraded by calpain (Goll et al., '91).

Furthermore, our calpain does not degrade tropomyosin as mammalian and avian calpains do. When we examined the effect of calpain on intact myofibrils we found that tropomyosin re- mains intact as well (unpublished data). This fact may be due to a possibly different conformation of frog tropomyosin from that of mammalian and avian tropomyosins.

It is now suggested that calpains are related to the myofibrillar protein turnover in both muscle and non-muscle cells, and this suggestion is supported by the fact that in various mus-

cular dystrophies high protein catabolism is ac- companied by increased calpain activity (Dayton et al., '81; Go11 et al., '91; Turner et al., '88; Yamamoto et al., '92). We believe that studies on the newly discovered tissue-specific calpains will give more direct evidence on the mechanism of the activation and the specificity of these proteases in vivo.

In conclusion, the results of the present study showed tha t calpain from the R . ridibunda skeletal muscle specifically leads to the complete removal of Z-lines from the isolated intact myofibrils, without any other apparent proteolytic degradation of their substituent proteins. Although calpain appears to degrade isolated G-actin and myosin from the same tissue of the animal in vitro, our results do not support the suggestion that this protease degrades those endogenous substrates in vivo. It is likely that calpain must be involved in very specific and strictly regulated processes of proteolytic modification rather than in a general, no-specific proteolysis into the living cells.

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