Isolation from porcine cardiac muscle of a Ca2+-activated protease that partially degrades myofibrils

of Molecular

Journal

Isolation

and Cellular

Cardiology

(1980)

12, 533-55

1

from Porcine Cardiac Muscle of a Ca2+-activated Protease that Partially Degrades Myofibrils

WILLIAM

R. DAYTON* of Animal

AND

JUDITH

V. SCHOLLMEYER

Science, University of hdinnesota, St. Paul, Minnesota 55108, U.S.A. tDepartment of Laboratory Medicine and Pathology, lJniversi& of Minnesota, Minneapolis, Minnesota 55427, U.S.A. *Department

(Received

20 March

1979, accepted in revised form)

R. DAYTON AND J. \'. SCHOLLMEYER. Isolation from Porcine Cardiac Muscle of a Gag+-Activated Protease that Partially Degrades Myofibrils. Journalof Moleculnr and Cellular Cardioloa (1980) 12, 533-551. A protein fraction displaying Cal+-activated proteolytic activity has been isolated from porcine cardiac muscle. The crude enzyme was purified approximately 2000 fold by isoelectric precipitation followed by gel permeation chromato&aphy and by ion exchange chromatography. The partially purified enzyme exhibited optimal activity against either cardiac myofibril or casein substrates between pH 7.5 and 8.0, and in the presence of 1 rnM Ca*+ and at least 2 mM Z-mercaptoethanol. The enzyme removes Z-discs from skeletal and cardiac myofibrils and also removes the density from intercalated discs of cardiac myofibrils. The enzyme hydrolyzes troponin-T and troponin-I of both cardiac and skeletal muscle myofibrils in &o. In its proteolytic effect on either cardiac or skeletal myofibrils and in all other propertics examined, the Ca2+-activated, cardiac protease is similar to a Ca*+-activated protease (CAF) recently purified from porcine skeletal muscle (Dayton, LV. R., Reville, L%‘. J., Gall, D. E. and Stromer, M. H. (1976) Biochemistry 15, 2159-2167). It is possible that the Ca2+-activated, cardiac protease plays a role in degradation of myofibrils in injured myocardial cells.

W.

I(EY

\L)ORDS:

Ca*+-activated

protease;

Myofibril;

Z-disc;

Intercalated

disc;

Proteolysis.

1. h-oduction In myocardial cell injuries induced by a variety of conditions including experimentally induced myocardial infarction [13, 201, ischemia [13, 201, C:a*+ overload [9, 241, isoproterenol treatment [2, 231, and hypovolemic shock [7, 141, the first ultrastructural changes observed in the contractile apparatus are often disruption of the intercalated disc, Z-disc and I-band of the myofibril [2, 7, 13, 14, 20, 23, 241. Although very little is known about the factor or factors causing these ultrastructural changes, it is probable that they result from enzymatic degradation of the affected myofibrillar structures by intracellular proteolytic enzymes. ,4n rndogenous cardiac muscle proteolytic enzyme(s) able to degrade the Z-disc, intercalated disc and I-band of cardiac myofibrils in vilro would thus be strongly 0022-2828/80/060533+ M.C.C.

19 $02.00/O

(0 1980 Academic

Prrss

Inc. (London)

1,imitc.d t

534

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SCHOLLMEYER

implicated in degradation of cardiac myofibrillar proteins during their physiological turnover and in disruption of intercalated discs, Z-discs and I-bands in sublethally injured or necrotic cardiac muscle. Recent reports have documented the presence of such an enzyme in skeletal muscle. This Ca2+-activated proteolytic enzyme (CAF) catalyzes in vitro degradation of Z-discs and I-bands in skeletal muscle myofibrils [3-5, 15, 171. The enzyme (CAF) is nonlysosomal [17], is optimally active at physiological pH and temperature [5], and has an obligatory requirement for Ca2+ as an activator [5]. The presence in cardiac muscle of an enzyme with catalytic properties similar to those of skeletal muscle CAF might well explain the degradation of Z-discs and I-bands of cardiac myofibrils in ischemic or necrotic cardiac muscle tissue. In addition, because the intercalated disc is a Z-disc analog containing cl-actinin [18] a cardiac form of CAF might be expected to degrade the intercalated disc. Thus, presence of an enzyme such as CAF in cardiac muscle tissue could explain much of the ultrastucturally observed degradation of myofibrils in myocardial injury. Consequently, the objectives of this study were to determine if an enzyme with catalytic properties analogous to skeletal muscle CAF is present in cardiac muscle tissue and to examine the effects of any such enzyme on the ultrastructural appearance and molecular conformation of cardiac myofibrils in vitro. 2. Materials Isolation

and

Methods

of the Ca2+-activated protease

Porcine hearts were obtained approximately 20 min after death and immediately cut open, rinsed of blood and immersed in ice. The left ventricle was removed, trimmed of excess fat and connective tissue, and ground. All subsequent steps in the isolation procedure were performed at 0-3°C and are similar to those previously described for isolation of skeletal muscle CAF [4]. The ground muscle was homogenized in a Waring Blender in 2.5 volumes (v/w) of a solution containing 50 mM Tris-acetate, pH 8.0, 4 mM EDTA. CAF was removed from the supernatant of this homogenization by isoelectric precipitation between pH 6.2 and 4.9. The precipitated CAF was redissolved and salted out at 40: ;) ammonium sulfate saturation. The protein fraction obtained in this way is referred to as the P 0--4,, CAF fraction. Preparation

of myofibrils

Rabbit skeletal and cardiac muscle myofibrils procedure of Go11 et al. [8] modified by omission Chromatographic Ultrogel AcA DEAE-cellulose

34 (LKB) (Whatman)

were prepared according to the of the Triton X-100 treatment.

procedures

was used for gel permeation was used for ion exchange

chromatography chromatography.

and Flow

CZi2+-ACTIVATED

CARDIAC

PROTEASE

DEGRADING

MYOFIBRILS

rates, column dimensions, buffer systems and protein loads for the columns in this study are given in the appropriate figure legends.

535 used

Sodium dodecyl sulfate gel electrophoresis Polyacrylamide done according cribed in detail

gel electrophoresis to the procedure elsewhere [4].

in the presence of sodium dodecyl sulfate was of Weber and Osborn [22] and has been des-

CAF activity assays In this study three different assays measuring, respectively, Ca2+-activated Z-disc removal, Ca2+-activated proteolytic activity, and Ca2+-activated myofibril degradation were used to monitor CAF activity [4, 51. (Assay procedures and controls have been described in detail previously [4].) The first assay utilized phase and electron microscopy to determine the ability of protein fractions to catalyze Ca2+-activated removal of Z-discs and intercalated discs from cardiac myofibrils in vitro [4]. The second assay routinely performed on cardiac CAF preparations used casein as a substrate to quantitatively measure Ca2+-activated proteolytic activity and has been described in detail elsewhere [4]. The third assay was devised to quantitate the Ca 2+-activated proteolytic release from myofibrils of material soluble in 100 mM KC1 and absorbing at 278 nm [4]. Throughout the study all fractions containing Ca 2+-activated Z-disc-removing activity were also found to contain Ca 2+-activated proteolytic activity against casein and against myofibrils. Additionally, no Z-disc-removing activity or proteolytic activity was observed in any fraction in the absence of Ca3+ (i.e.. presence of 5 mM EDTA).

Ca2+ dependence of CAF activity Two separate studies, one using casein as a substrate and the other using cardiac myofibrils as a substrate, were done to determine the effect of C1a2+ concentration on CAF activity. Procedures for both casein and myofibril assays were identical to those already described [5].

pH dependence

qf CAF

activity

The pH dependence of Caz+activated protelytic activity isolated from cardiac muscle was determined using cardiac myofibrils as a substrate. C:AF activity was assayed at pH values between 3.0 and 9.5 at 25°C for 30 min, according to procedures described in detail elsewhere [S]. Y2

536

W.

R.

DAYTON

Temperature

AND

J. V.

SCHOLLMEYER

dependence of CAF activity

Studies on temperature dependence were done using casein as a substrate. Assay procedures and conditions were identical to those previously described [5]. E$ect of Z-mercaptoethanol

concentration on CAF activity

Effect of 2-mercaptoethanol concentration on CAF activity either myofibrils or casein as a substrate. Assay procedures identical with those previously described [5].

was determined and conditions

using were

CAF treatment of skeletal and of cardiac myofibrils Both skeletal and cardiac myofibrils were treated with CAF and subsequently prepared for SDS polyacrylamide disc gel electrophoresis according to procedures described in detail previously [3]. Specific details of individual CAF treatments done in this study are given in the appropriate figure legends. Phase and electron microscopy Aliquots of all the myofibrillar samples, both experimentals and controls, were routinely examined by phase microscopy (Zeiss Universal, 100X Planachromat objective) in order to determine the extent of Z-disc removal. Myofibrillar samples of all the treatments were also taken for electron microscopic analysis. EM samples were then processed in the following manner. They were pelleted at 700 xgmax for 10 min (room temperature) in a bench-top centrifuge. Cold 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, was layered gently over the pellet and the pellet was allowed to stand undisturbed for 10 min. (This step and all subsequent steps prior to addition of the embedding resin were carried out on ice.) After 10 min the pellet was gently dislodged from the conical centrifuge tube, sliced into small cubes (1 mm) with a razor blade, and fixation of the pellet cubes continued for 1.5 h with several changes of glutaraldehyde. After a 15 min rinse in 0.1 M cacodylate buffer, pH 7.2, with several changes, the samples were postfixed in 1 y0 osmium tetroxide, 0.1 M cacodylate buffer, pH 7.2, for 1 h. Following a dehydration series of graded acetones, the samples were embedded in an EponAraldite mixture [I]. Thin sections of these samples which had been routinely stained with uranyl acetate and lead citrate were viewed in a Philips 300 electron microscope. 3. Results Isolation

and partial

purijcation

of a CAF-like

enzyme from porcine cardiac muscle

Using the CAF isolation procedure described in the Methods Section, it was possible to isolate from porcine cardiac muscle a P 0--40 protein fraction containing

Ca2+-ACTIVATED

CARDIAC

PROTEASE

DEGRADING

537

MYOFIBRILS

Ca2+-activated proteolytic activity and Ca2+ -activated Z-disc-removing activity. The P,-,, fraction containing presumptive CAF activity was then subjected to Ultrogel AcA 34 gel permeation chromatography. All of the Ca2+-activated Z-disc-removing activity and Ca2+ -activated proteolytic activity in the PO_,,, fraction eluted together as a single peak from the Ultrogel column [Figure 1 (a)]. The Ca2+-activated proteolytic and Z-disc-removing activities obtained from the cardiac P,,--4O fraction eluted identically to CAF activity obtained from a skeletal muscle P,-,, fraction. The specific Ca 2+-activated proteolytic activity and the specific myofibril degrading activity of the cardiac CAF fraction obtained from the Ultrogel columns was 3 to 4 times greater than that of the cardiac POeJO fraction. The Ultrogel AcA 34 fraction containing Ca 2+-activated Z-disc-removing

-

5.0

0.8 0.6 0.4 0.2 0

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

1” 30 25 20 15 IO 5

74 Fraction

number

0

400

I 300

$I

200 100

=: El

0

FIGURE 1. (a) Elution profile of the P,_,, CAF fraction from a 2.5 x 80 cm Ultrogel AcA 34 column. The column was loaded with 200 mg CAF protein in 5 ml and was &ted with 20 msr Tris-acetate, pH 7.5, 1 mM EDTA, and 1 msr NaN, at 15 ml/h. Seven-milliliter fractions were collected. (---) specific Ca a+-activated proteolytic activity determined used casein as a substrate; (.-) absorbance at 280 nm. The fraction containing Ca rf-activated proteolytic activity against casein also contains Car+-activated Z-disc-removing activity. The fraction indicated by the vertical lines was used in the next purification step. (b) Elution profile of Ultrogel AcA 34 purified CAF from a 1.4 x 10 cm DEAE-cellulose column. The column was loaded with 40 mg of protein in 55 ml and was eluted with a continuous gradient consisting of 50 ml each of 150 mM KCl, 20 mM Tris-acetate, pH 7.5, 1 mM EDTA, 0.1 “/b 2-mercaptoethanol, and of 350 rn~ KCl, 20 mM Trisacetate, pH 7.5, 1 mr.r EDTA, 0.19; 2-mercaptoethanol. Flow rate was 10 ml/h and 5.0 ml fractions were collected. (-.-.-) KC1 concentration in eluant; (---) specific Cal+-activated proteolytic activity determined by using casein as a substrate; (--) absorbance at 280 nm. The fraction containing Ca*+-activated proteolytic activity was the only fraction containing CaZ+activated, Z-disc-removing activity. The indicated fraction was used in experiments on properties of cardiac CAF.

I

538

W.

R. DAYTON

AND

J. V. SCHOLLMEYER

activity and Ca2+-activated proteolytic activity was adjusted to a final KC1 concentration of 150 mM and to a final 2-mercaptoethanol concentration of 0.1% and was loaded directly onto a DEAE-cellulose column. Ca2+-activated Z-disc-removing activity eluted together proteolytic activity and Ca 2+-activated from this column as a peak between 230 and 280 mM KC1 [Figure 1 (b)]. This is exactly where skeletal muscle CAF elutes from similar DEAE-cellulose columns [4]. DEAE-cellulose chromatography routinely resulted in a nine- to proteolytic and Z-disc-removing tenfold increase in the specific Ca2+- activated activities. Thus, Ca2+-activated proteolytic activity and Caz+-activated Z-discremoving activity co-chromatograph in both gel permeation (Ultrogel AcA 34) and ion exchange (DEAE-cellulose) columns. Additionally, the cardiac, CazA-activated proteolytic and Z-disc-removing activities elute from both of these columns identically to skeletal muscle CAF. Because Ca2+-activated proteolytic activity and Ca2+-activated Z-disc-removing activity can be isolated from cardiac muscle and because these activities elute together from a gel permeation column and a subsequent DEAE-cellulose column at the same elution volume and ionic strength, respectively, at which skeletal muscle CAF elutes, we believe that these activities are due to a cardiac form of CAF. Efect of Ca2f on cardiac CAF activity Optimal Ca2+ concentration for CAF catalyzed hydrolysis of myofibrils is 1.0 mM (Figure 2) and CAF appears to be inactive below 0.1 mM Ca2+. Calcium concentrations of 10 mM consistently inhibit CAF’s degradation of myofibrils, however the reason for this inhibition is unclear. The Ca2+ requirement for CAF-catalyzed proteolysis of casein was identical to the Ca2+ requirement for CAF-catalyzed degradation of myofibrils, suggesting strongly that both Ca2+-activated proteolysis

mM Ca2+ FIGURE 2. Effect of Ca2+ concentration on rate cardiac myofibrils by cardiac CAF. Assay conditions: 10 rn~ Z-mercaptoethanol, Ca2+ as indicated, 2.5 mg cellulose-purified CAF/ml. 30 min, 25”C, 2.0 ml fmal

of release of soluble peptides from porcine 100 rn~ KCI, 100 rn~ T&acetate, pH 7.5, of myofibrillar protein/ml, 15 pg of DEAEvolume.

Ca2+-ACTIVATED

CARDIAC

PROTEASE

DEGRADING

MYOFIBRILS

539

of casein and Ca2+-activated degradation of myofibrils are caused by a single Ca2+-activated protease. In addition, the response of cardiac CAF activity to Ca2+ concentration was identical to the response of skeletal muscle CAF to ca2+ [5]. pH optimum of cardiac CAF While the pH optimum for cardiac CAF activity was between 7.5 and 8.0, significant CAF activity was detected between pH 6.0 and 8.5. Activity decreased rapidly below pH 6.0 or above pH 8.5. This response of cardiac CAF activity to pH, makes it very unlikely that CAF originates from lysosomes (where the internal pH is thought to be in the acidic range). As was the case with the Ca2+ requirement for cardiac CAF activity, the response of cardiac CAF to pH is very similar to the response of skeletal CAF [5].

Ejkt

of 2mercaptoethanol

and temperature on cardiac CAF activity

Ciardiac CAF requires a reducing agent in order to function optimally. Approximately 2 mM P-mercaptoethanol is sufficient to activate the enzyme maximally. Skeletal muscle CAF requires presence of a similar level of 2-mercaptoethanol for optimum activity [5]. Studies of cardiac CAF activity against casein at 25” and 37.5” show significant differences in the activity and stability of the enzyme at these temperatures. A 25°C: CAF activity remains constant for up to 1 h. At 37.5” the initial rate of casein hydrolysis by CAF is more rapid than at 25”C, however the enzyme rapidly becomes inactivated after 10 min at 37.5”C is completely inactive. This response of cardiac CAF activity to temperature is identical to the response of skeletal muscle CAF activity at the same assay temperature [5].

Phase and electron microscopy It has previously been shown that in vitro treatment of skeletal myofibrils with purified skeletal muscle CAF causes removal of Z-discs [3-51. In the present study, phase microscopic results show that treatment of skeletal myofibrils with the protease (0.1 mg Ca2+-activated DEAE-cellulose-purified, cardiac Ca s+-activated protease/lO.O mg skeletal myofibrils) also caused rapid and dramatic Z-disc removal [Figure 3(a)] while the control myofibrils,demonstrated healthy Z-discs [Figure 3(b)]. In cardia CAF-treated myofibrils in which Z-discs could no \ longer be detected using phase microscopy [Figure 3(a)] the average length of a myofibril fragment (5 sarcomeres) was shorter than in control myofibrils ( 10 sarcomeres) in which Z-discs were intact. This increased fragmentation of skeletal myofibrils following cardiac CAF treatment suggests that cardiac CAF catalyzes

540

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R.

DAYTON

AND

J.

V.

SCHOLLMEYER

FIGURE 3. Phase micrographs of rabbit skeletal myofibrils preparrd according to the method of Go11 et al. [IS]. x 160. (a) Myofibril incubated for 1 h at 25-C in the presence of 100 rn~ KCl, 50 rn~ Tris-acetate, pH 7.5, 5 rn~ 2-mercaptoethanol, 5 rn~ Ca2 ‘, 1 rn~ NaN,, 0.05 mg of CAF/ml. Z-discs are removed. (b) Myofibril incubated for 1 h at 25’C in which 10 rn~ EDT.4 is substituted for 5 rnM Ca’+ in the above incubation mixture. Z-discs (arrows) are obviously present.

removal of enough structural material from the Z-disc to allow the two halves of the I-band to separate. This suggestion gains credibility upon examining the effect of cardiac CAF treatment on the ultrastructure of skeletal myofibrils (Figure 4). Cardiac CAF-treated myofibrils show extensive Z-disc disintegration [Figure 4(a)] while control myofibrils display little or not ultrastructural changes [Figure 4(b)]. In addition, thin filaments in cardiac-CAF-treated myofibrils are not as well organized relative to each other as are thin filaments in control myofibrils. Many thin filaments in the treated myofibrils are oriented transversely to the longitudinal axis of the myofibril. Whether this lack of thin filament organization in the treated myofibrils is the result of CAF’s direct effect on the thin filaments or whether it is the result of loss of the Z-disc as an attachment point for the thin filament is not clear from the present data. Our data does show, however, that treatment ofskeletal muscle myofibrils with the Ca’T-activated, cardiac protease results in the same changes in myofibrillar ultrastructure as does treatment of myofibrils with purified skeletal CAF [5]. Cardiac myofibrils treated with the C:a2~--activated, cardiac protease also display disintegration of the Z;disc region and disorganization and possible degradation of the thin filaments in the I-band [Figure 5(a)]. Again, no significant ultrastructural changes are seen in the control cardiac myofibril [Figure 5(b)].

Ca2+-ACTIVATED

FIGURE > 12 800. described associated described

CARDIAC

PROTEASE

DEGRADING

MYOFIBRILS

541

4. Electron micrographs of longitudinally-oriented rabbit skeletal myofibrilh. (a) Micrograph represents myofibrils incubated with C4F in a manner identical to that in 3(a). All that appears to remain of the Z-disc are small regions of density iarrow\) with the I-filaments. (b) Micrograph represents the control myofibrils inrubared a\ in 3(b). Z-discs are obviously intact.

The effect of the cardiac, Ca2+- activated protease on the intercalated disc was of prime interest in this study since we have determined by other methods [ 18 1 that the protein composition of the intercalated disc is similar to that of the Z-disc. As demonstrated in Figure 6(a), the cardiac, Ca 2+-activated portease removes a substantial amount of dense material from the intercalated disc. In addition, I-filaments which are adjacent to the intercalated disc appear either to have detached from it or to have coalesced around it. An intercalated disc displaying the normal amount of density and I-filament juxaposition is seen in the micrograph of a control myofibril [Figure 6(b)].

542

W.

R. DAYTON

AND

J. V.

SCHOLLMEYER

FIGURE 5. Electron micrographs of longitudinally-oriented rabbit cardiac myofibrils. x 14 880. (a) CAF-treated myofibril as in 3(a). This myofibril demonstrates some contraction such that I-filaments are not readily discernible in the I-band (I) region. No Z-discs remain. (b) Control myofibril treated as in 3(b). Again this myofibril demonstrates some degree of contraction, however, the large Z-discs (arrows) characteristic of cardiac muscle are easily seen.

E$ect The (myosin,

of

effects actin,

of

cardiac and skeletal CAF on the myojibrillar myojbrils in vitro purified a-actinin,

skeletal troponin,

muscle

CAF

tropomyosin

on and

proteins on skeletal and

purified C-protein)

myofibrillar and

proteins on myofibrils

Ca2+-ACTIVATED

CARDIAC

PROTEASE

DEGRADING

MYOFIBRILS

FIGURE 6. Electron micrographs of intercalated discs (ICD) of rabbit cardiac .’ 34 400. (a) CAF-treated intercalated disc as in 3(a). ICD displays loss of dense material opposmg cell membranes (arrows) while (b) the control ICD treated as in 3(b) retains amorphous material which is normally seen adjacent to the opposed cell membranes.

543

myofibrils. near the the dense,

from skeletal muscle are quite specific and have been described previously [3]. Skeletal CAF hydroylyzes troponin-T, troponin-I, and tropomyosin. Additionally, it cleaves C-protein, reducing its molecular weight by approximately 10 000 daltons [3]. Skeletal CAF has no detectable effect on myosin, actin or a-actinin 131. Because the effect of skeletal CAF on skeletal myofibrils is well characterized,

544

W.

R.

DAYTON

AND

J.

V.

SCHOLLMEYER

we felt that in assessing the similarities between activated proteases it would be useful to compare skeletal myofibrils. The electrophoretic banding with either purified skeletal CAF [Figure 7(c)] cardiac protease [Figure 7(d)] are identical. (a)

(b)

the skeletal and cardiac Cat+the effect of these proteases on pattern of skeletal myofibrils or the DEAE-cellulose-purified (cl

(d)

Ins.-

Actin37KTM/ 30K24K-

--m

FIGURE 7. Seven and one half percent, SDS polyacrylamide gels of rabbit skeletal myofibrils before and after treatment with either skeletal or cardiac CAF. All gels are loaded with 80 pg protein. (a) Control skeletal myofibrils incubated for 60 min at 25°C in 100 rnM KCI, 50 mM Tris-acetate, pH 7.5, 5 rnM CaCl,, 10 rnM Z-mercaptoethanol, 1 mM NaN,. (b) Control skeletal myofibrils incubated for 50 min at 25°C in 100 rnM KCl, 50 mM Tris-acetate, pH 7.5, 10 mM 0.1 mg DEAE-purified, cardiac CAF/lO mg EDTA, 10 mM Z-mercaptoethanol, 1 mM NaN,. myofibrillar protein. (c) Skeletal myofibrils incubated for 60 min at 25°C in 100 rnM KCl, 50 mM Tris-acetate, pH 7.5, 5 rnM CaCl,, 10 mM Z-mercaptoethanol, 1 rnM NaN,, 0.01 mg purified skeletal CAF/lO mg myolibrillar protein. (d) Skeletal myofibrils incubated for 60 min at 25°C in 100 mM KCI, 50 rnM Tris-acetate, pH 7.5, 5 rnM CaCl,, 10 rnM 2-mercaptoethanol, 1 rnM NaN,, 0.1 mg DEAE-purified, cardiac CAF/ 10 mg myofibrillar protein.

Proteolytic breakdown products are seen at 130 000 daltons (one of these is the result of CAF’s action on C-protein ( 140 OOO-daltons) [II] and at approximately 100 000 daltons. Although the origin of all but one of these breakdown products is unclear, it is unlikely that they originate from either myosin or m-protein since skeletal CAF has previously been shown to be inaffective against these proteins. It is significant to note that our myofibril preparations contain a small amount of proteinaceous material (possibly connective tissue) that is insoluble under the

Ca2+-ACTIVATED

CARDIAC

PROTEASE

DEGRADING

MYOFIBRILS

545

conditions used for SDS electrophoresis and that consequently barely enters the gel. The appearance of a new band slightly below the top of the gel in the electrophoretic banding pattern of myofibrils treated with either skeletal CAF [Figure 7(c)] or the cardiac protease [Figure 7(d)] indicates that both enzymes are hydrolyzing these insoluble proteins and it is thus possible that the unidentified breakdown products at 130 000 and 100 000 daltons originate from this hydrolysis. Breakdown products present at 30 000 and 14 000 daltons in protease treated myofibrils are identical in molecular weight to products formed by skeletal CAF’s hydrolysis of skeletal troponin-T and troponin-I, respectively [3]. Consistent with the ability of both proteases to remove Z-discs from skeletal myofibrils, treatment of myofibrils with either enzyme causes a substantial lessening in density of the 96 OOO-dalton band corresponding to u-actinin [19]. Treatment of skeletal myofibrils with either protease also results in hydrolysis of troponin-T (37 000 daltons) [6] and troponin-I (24 000 daltons) [6] as evidenced by the absence of bands corresponding to these polypeptides in gels of treated myofibrils [Figures 7(c) and 7(d)]. Tropomyosin in the myofibril does not appear to undergo significant degradation by either protease under the conditions used in this study. More extensive CAF treatment of myofibrils has been shown to cause breakdown of tropomyosin, however [6]. Because the electrophoretic banding patterns of skeletal myofibrils treated with either skeletal CAF or the cardiac protease are identical, it appears that the effects of the cardiac, Ca2+-activated protease on myofibrillar proteins of skeletal myofibrils are identical to the effects of skeletal CAF on these proteins. The electrophoretic banding pattern of cardiac myofibrils treated in vitro with either purified skeletal CAF [Figure 8(c)] or the DEAE-cellulose-purified, Ca2+-activated, cardiac protease [Figure 8(d)] are also identical, thus indicating that the two proteases are identical in their ability to hydrolyze specific proteins Identification of specific proteins found in the cardiac myofibril preparation. affected by the proteases is difficult since the electrophoretic banding pattern of cardiac myofibrils is not as well characterized as that of skeletal myofibrils and since the effect of purified skeletal CAF on purified cardiac myofibrillar proteins has not been examined. Consequently, in order to unequivocally identify cardiac myofibrillar proteins affected by CAF it will ultimately be necessary to examine the effect of the purified protease on purified cardiac myofibrillar proteins. Even so, we can draw some conclusions concerning the effect of either skeletal CAF or the cardiac protease on cardiac myofibrillar proteins by examining the electrophoretic banding pattern of cardiac myofibrils treated with one of these enzymes. Treatment of cardiac myofibrils with either protease has little if any detectable effect upon the heavy chains of myosin or upon actin since no detectable differences exist between the bands corresponding to these polypeptides in the electrophoretic banding pattern of control [Figure 8(a), (b)] and protease treated

546

W.

R.

DAYTON

AND

(0)

J.

V. (b)

SCHOLLMEYER Cc)

(d)

Ins.-

Actin“T”lF 27.5K22K-

FIGURE 8. Seven and one half percent, SDS polyacrylamide gels of rabbit cardiac myofibrils before and after treatment with either skeletal or cardiac CAF. All gels are loaded with 80 yg of protein. (a) Control cardiac myofibrils incubated for 60 min at 25°C in 100 rnM KCI, 50 mM Tris-acetate, pH 7.5, 5 rnM CaCl,, 10 rnM Z-mercaptoethanol, 1 rnM NaN,. (b) Control cardiac myofibrils incubated for 60 min at 25°C in 100 rnM KCI, 50 mM Tris-acetate, pH 7.5, 10 rnM EDTA, 10 rnM 2-mercaptoethanol, 1 mM NaN,, 0.1 mg DEAE-purified cardiac CAF/lO mg myofibrillar protein. (c) Cardiac myofibrils incubated for 60 min at 25°C in 100 rnM KCl, 50 rnM Tris-acetate, pH 7.5, 5 mM CaCl, 10 rnM 2-mercaptoethanol, 1 rnM NaN, 0.01 mg purified skeletal CAF/lO mg myofibrillar protein. (d) Cardiac myofibrils incubated for 60 min at 25°C in 100 mM KCI, 50 mM Tris-acetate, pH 7.5, 5 rnM CaCI,, 10 rnM 2-mercaptoethanol, 1 rnM NaN,, 0.1 mg DEAE-purified, cardiac CAF/lO mg myofibrillar protein.

[Figure 8(c), (d)] myofibrils. The density of a 96 OOO-dalton band (a-actinin) [IO] is substantially diminished, however, in the electrophoretic banding pattern of myofibrils that have been treated with either protease. This result is not surprising since both proteases degrade the Z-disc and u-actinin is a major component of this structure [18]. Additionally, the band corresponding to troponin-T (40 000 daltons) [IO] is completely removed and a band at 27 500 daltons is substantially diminished in density [Figure 8(c), (d)] by treatment of myofibrils with either protease. Because the 27 500-dalton band is composed of both troponin-I (28 000 daltons) [IO] and LC, of myosin (27 OOO-daltons) [IO], it is

CB2+-ACTIVATED

CARDIAC

PROTEASE

DEGRADING

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not possible to state unequivocally from our data whether troponin-I (TN-I) or LC, or both are being degraded by CAF. Since both skeletal CAF and cardiac CAF degrade TN-I in skeletal myofibrils and have no detectable effect on LC,, it is likely that these proteases also degrade TN-I in cardiac myofibrils and that the density remaining in 27 500-dalton band after protease treatment is due to the presence of LC,. This conclusion is supported by the fact that treatment of IZI brushes (a preparation of Z-discs and attached thin filaments) with either protease results in complete loss of the 27 500-dalton band (unpublished observation). Since myosin has been extracted from IZI brushes, the 27 500-dalton band present in their electrophoretic banding pattern is composed almost completely of troponin-I and since treatment of IZI brushes with either protease causes complete removal of this band, it seems that cardiac troponin-I is very susceptible to degradation by either proteolytic enzyme. Consequently, it is likely that the reduction in density of the 27 500-dalton band upon CAF treatment of cardiac myofibrils is due to hydrolysis of its troponin-I component and that the density remaining after CAF treatment is due to the presence of LC, of myosin. In addition to the previously mentioned changes in the electrophoretic banding pattern of cardiac myofibrils upon treatment with either skeletal or cardiac CAF, we also noted the appearance of apparent proteolytic breakdown products banding at approximately 130 000, 104 000 and 22 000 daltons [Figure 8(c), (d)]. Although the origin of these breakdown products is not known it is possible that at least some of them arise from hydrolysis of proteinaceous material banding at the very top of the gel. As is the case in protease treated skeletal myofibrils, the presence of a band immediately below this material in the banding pattern of protease treated myofibrils indicates that some of these proteins that do not enter the gel are being hydrolyzed. 4. Discussion This study describes the isolation and partial purification from cardiac muscle of a Ca2*-activated proteolytic enzyme whose properties are similar to those of a Ca2+.-activated protease (CAF) hypothesized to be involved in degradation of myofibrillar proteins in skeletal muscle [3 to 51. The cardiac protease elutes from both gel permeation and ion exchange columns at the same point as skeletal muscle CAF, has the same effect on skeletal and cardiac muscle myofibrils and has the same Ca2+, pH and temperature optimum as skeletal muscle CAF, and is isolated from cardiac muscle via a procedure very similar to the procedure used to isolate CAF from skeletal muscle. It thus seems quite likely that this Ca’+activated protease is the cardiac form of the CAF enzyme previously described in skeletal muscle [3 to 51. Although in vitro studies show conclusively that cardiac CAF degrades both Z-discs and intercalated discs the mechanism of this degradation is unclear,

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partially because of uncertainties about the molecular structure and protein composition of these structures. The electron dense material removed from the Z-disc and intercalated disc regions rather rapidly by cardiac (:AF is almost certainly composed partly of a-actinin since removal of this substance coincides with appearance of a 96 OOO-dalton peptide in supernatants from CAF-treated myofibrils (unpublished observation). This finding is consistent with antibody localization of a-actinin in the dense region of both Z-discs and intercalated discs [ 181. The ability of cardiac CAF to degrade intercalated discs and partially degrade myofibrils appears to be unique, since, to our knowledge, no other cardiac protease has been shown to degrade myofibrils at the neutral pH found in the cytoplasm. Although the physiological role of cardiac CAF is not yet known, the fact that CAF is the first enzyme isolated from cardiac muscle cells that is capable of degrading intact myofibrils at neutral pH suggests that the enzyme may play a role in the rapid degradation of myofibrillar proteins observed in injured cardiac muscle tissue. This hypothesis seems even more feasible when one notes that in many types of myocardial injury the initial alterations in myofibrillar ultrastructure are degradation of the Z-disc, intercalated disc and I-band [Z, 7, 13, 14, 20, 23, 241 all of which could be caused by cardiac CAF. Although CAF may be involved in the initial degradation of myofibrils, the inability of CAF to degrade the two major muscle proteins, actin and myosin, suggests that other proteolytic enzymes are necessary to completely disassemble the myofibrillar structure. The identity of these additional proteases is not yet known, however, lysosomal cathepsins would be prime candidates. Our studies indicate that cardiac CAF is active in the pH range between 5.5 and 9.5 and at Ca2+ concentrations between 0.1 mM and 1 mM. CAF is maximally active in the neutral pH range maintained by a healthy cell, however the enzyme retains significant activity at the lower pH’s which might be presented in cardiac muscle cells during anoxia or ischemia [16]. Thus it seems that the pH requirements for CAF activity are consistent with the hypothesized role of the enzyme in both metabolic turnover of myofibrillar proteins in normal cardiac cells and rapid degradation of myofibrillar proteins in injured cardiac muscle cells. On the other hand, the in vitro Ca2+ requirement for CAF activity is at least an order of magnitude higher than the level of free Ca2+ thought to be present in healthy cardiac muscle cells and initially might be interpreted as precluding CAF from any role in degradation of myofibrillar protein in these cells. It is possible, however, that Ca2+ levels required to activate CAF in vitro are higher than those required in vivo. It is also of interest that several studies have shown that in certain cardiac myopathies (e.g. those caused by experimentally induced myocardial infarction [13, 201, ischemia [13, 201, Ca2+ overload [9, 241 or isoproterenol treatment [2, 211) involving degradation of the Z-disc and I-band of the myofibril, the ability of the sarcoplasmic reticulum and sarcolemma to control Ca”+ flux

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into the cardiac muscle cell is impaired. This impairment presumably results in increased movement of Caa+ into the cell and increased intracellular levels of free Ca2+. It is possible that the increased influx of Ca2+ activates CAF in the cardiac muscle cell, triggering the observed degradation of intercalated discs, Z-discs and I-bands. Thus there is evidence that some kinds of cardiac necrosis are caused by or accompanied by increased levels of intracellular free Ca2+ that might be sufficient to activate cardiac CAF. Future studies will investigate the level of CAF in some of these myopathies and attempt to ascertain the nature of the possible interaction between intracellular Ca2+ and CAF in necrotic cardiac muscle cells. In addition to Ca2+ concentration, in vivo CAF activity may be regulated by the presence of an endogenous inhibitor of CAF activity in cardiac muscle. Such an inhibitor of CAF has been isolated and partially purified from skeletal muscle [12] and cardiac muscle [21]. We have also isolated a similar inhibitor of CAF activity from cardiac muscle and have found it to be very effective in inhibiting CAF activity in vitro. However, further experimentation is necessary to determine whether this inhibitor functions to regulate CAF activity in vivo. Because degradation of Z-discs, intercalated discs and thin filaments is the first ultrastructure change observed in myofibrils from cardiac muscle injured by a variety of procedures and because CAF degrades or partially degrades all of these structures in vitro, we believe that the presence of the enzyme in cardiac muscle is significant. Results presented in this study show that CAF or a CAF-like enzyme is present in cardiac muscle and has the ability to catalyze the initial degradative changes observed in myofibrils from injured myocardial cells. It is thus possible that CAF is the factor responsible for initiating myofibrillar breakdown observed in injured myocardial tissue. We are presently in the process of studying the location of CAF and controls on its activity in an effort to elucidate the exact role of the enzyme in myofibril degradation.

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