Dissociation of myosin light chains and decreased myosin ATPase activity with acidification of synthetic myosin filaments: Possible clues to the fate of myosin in myocardial ischemia and infarction

Journal

of Molecular

and Cellular

Cardiology

(1980)

12,

149-164

Dissociation of Myosin Light Chains and Decreased Myosin ATPase Activity with Acidification of Synthetic Myosin Filaments: Possible Clues to the Fate of Myosin in Myocardial Ischemia and Infarction THOMAS

C. SMITHERMAN, DALE E. GLEN RICHARDS

W. DYCUS

AND

The Medical and Pre-Clinical Science Services, VA Medical Center and the Departments of Medicine and Biochemistry, Southwestern Medical School, The University of Texas Health Science Center at Dallas, 4500 S. Lancaster Road, Dallas, Texas 75216 U.S.A. (Received

30 January

1979,

accepted in revisedform

2 1 June

1979)

T. C. SMITHERMAN, D. W. DYCUS AND E. G. RICHARDS. Dissociation of Myosin Light Chains and Decreased Myosin ATPase Activity with Acidification of Synthetic Myosin Filaments: Possible Clues to the Fate of Myosin in Myocardial Ischemia and Infarction. j%urnal of Molecular and Cellular Cardiology (1980) 12, 149-l 64. The effect of mild acidification of synthetic (reconstituted) myosin filaments was studied in order to gain insight into some of the possible effects of ischemia-induced intracellular acidosis on the structure and function of myosin following myocardial infarction and myocardial ischemia. Degradation products of myosin that are soluble (at physiologic ionic strength and pH) would be of potential diagnostic value for myocardial infarction. Acidification of rabbit skeletal synthetic myosin filaments led to a pH dependent partial dissociation of the heaviest (LC,) and lightest (LC,) of the 3 light chains. Dissociation was detected from pH 5.0 to 6.5 and was maximal at pH 6.0, at which 30:; of LC, was dissociated. Acidification of canine cardiac synthetic myosin filaments led to partial dissociation of both light chains; but more LC, than LC, was dissociated. Light chains reassociated with heavy chains upon return of the pH to 7. Light chains of myosin have recently been reported to appear in the peripheral blood after myocardial infarction but the small amount of free light chains in the heart is insufficient to account for the amount that appears in the blood. Acid-mediated dissociation of light chains in vitro suggests that circulating light chains after myocardial infarction may arise as a result of the intracellular acidosis of ischemir myocytes. The mechanisms responsible for the acidification-induced decrease in myofibrillar actomyosin adenosine triphosphatase (ATPase) activity are unclear. One possibility is that the decreased myofibrillar ATPase activity is due in part to an acidinduced decrease of the myosin ATPase of the myofibril irrespective of the effect of acid on the troponin-tropomyosin regulatory system. This possible mechanism is supported by the observations that acidification of rabbit skeletal and human and canine cardiac synthetic myosin filaments resulted in a reduction of iZTPase activity (measured at pH 7.5) of the redissolved myosin which was progressive with greater acidification. The reduction in ATPase activity occurred whether the return of the myosin to pH 7.5 was accomplished in the presence or absence of dissociated light chains. Supported by USPHS [HLl7669 American Heart 002%2828/80/020149+ M.C.C.

the

Research (Ischemic Association,

Service of the \.ctrrans Administration Heart Disease SCOR), and HL149381, and the Moss Heart Fund, Dallas, Texas,

15 $02.00/O

%‘ 1980

Academic

and by the Texas U.S.A. Press

Inc.

grants from the Affiliate of the (London)

Limited F

150

T. C. SMITHERMAN KEY WORDS: Myocardial Dissociation of myosin; ischemia.

infarction; Myosin light

ET AL.

Myocardial ischemia; chains; Intracellular

Myosin Acidosis

ATPase activity; with myocardial

1. Introduction Relatively little is known about the fate of myosin following myocardial infarction and severe myocardial &hernia. The classic light microscopic studies by Mallory et al. [23] and Lodge-Patch [ZO] of the heart after myocardial infarction in man demonstrated that removal of muscle fibers is relatively slow, beginning about 6 h after infarction and continuing for 2 to 6 weeks. While it has been reported that myosin with an intact adenosine triphosphatase (ATPase) activity can be extracted from infarcted muscle for as long as one month after acute infarction [5] it has also been noted that there is a marked decrease in the amount of myosin in the infarcted zone within 24 to 48 h of the infarct [II]. Because of the large amount of myosin in the myocyte and because of its unique solubility properties, it is of interest to determine whether degradation of myosin in the myocyte following infarction is associated with identifiable, soluble (at physiologic ionic strength and pH) products of myosin that could cross the damaged sarcolemma and appear in the peripheral circulation. Such degradation products would be of potential diagnostic value for myocardial infarction. It has been reported recently that myosin light chains appear in the peripheral blood after myocardial infarction in men [33] and experimental animals [18]. The small amount of free light chains in the heart is insufficient to account for the amount that appears in the blood [7]. A mong the potential causes of,ischemiainduced cell damage is the acute intracellular acidosis that occurs aft& coronary artery occlusion [1.5, 341. Therefore, one of the purposes of the present study was to determine if mild acidification of synthetic (reconstituted) myosin filaments caused dissociation of the light chains of myosin from heavy chains, suggesting a possible source of the circulating light chains after myocardial infarction. Tennant & Wiggers suggested in 1935 that the decreased contractility of acutely ischemic myocardium was the consequence of an acidosis-mediated decrease in the intropic state [32]. I n recent years, considerable interest has been given to possible acidosis-mediated intracellular defects that may contribute to this “early pump failure” of acute myocardial ischemia [15, 341. Among these possible defects is an acidosis-mediated decline in actomyosin ATPase activity. This ATPase activity occupies a central position in the transduction of chemical to mechanical energy. Any ischemia-induced changes in the contractile proteins that would reduce actomyosin ATPase activity would decrease the velocity of cardiac contraction. A decrease in actomyosin ATPase activity with decreasing pH has been demonstrated with in vitro experiments with both skeletal [17, 25, 281 and cardiac [17, 381 myofibrils. The reasons for this acidosis-mediated decrease in ATPase

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remain unclear. One of the several possibilities that have been considered, that acidification may alter the myosin ATPase of the myofibril irrespective of the effect on the troponin-tropomyosin regulatory complex, has not been extensively investigated. Accordingly a second purpose of the present study was to investigate the effect of acidification of synthetic myosin filaments on myosin ATPase. 2. Materials Preparation

and

Methods

of myosin

Rabbit white skeletal myosin Rabbits were killed by a blow to the neck. White skeletal myosin was prepared from the backstrap muscles as described earlier [27] except that QAE-Sephadex chromatography instead of DEAE-Sephadex was utilized for final column purification. Canine and human cardiac myosin Dogs were killed by intravenous barbiturate injection. The hearts were immediately removed and chilled. Human hearts were obtained following forensic autopsy within 24 h of death and were immediately frozen and maintained at -60” to -20°C until used. All subsequent steps were performed at 0 to 4°C. Left ventricular myocardium was isolated, weighed, and minced. To remove proteins soluble at low ionic strength, the myocardium was thrice blended with 5 volumes of buffer consisting of 0.05 M KPO,, 0.01 M Na pyrophosphate, 0.001 M EDTA and 0.002 M dithiothreitol (DTT), pH 6.8, for 10 set of each min. for 10 min followed by centrifugation at 10 000 g for 10 min. Myosin was extracted from the pellet for 25 min in a modified Guba-Straub buffer, pH 6.8, containing 0.3 M KCl, 0.15 M KPO,, 0.01 M Na pyrophosphate, 0.001 M EDTA, and 0.002 M DTT. The supernatant solution remaining after centrifugation for 10 min at 10 000 g was filtered through gauze and diluted with 10 volumes of cold buffer containing 0.001 M EDTA and 0.01 M Na pyrophosphate, pH 7.0. The myosin pellet collected by centrifugation at 27 000 g for 15 min was taken up in a minimum amount of buffer at pH 6.8 containing 0.5 M KCl, 0.02 M Na pyrophosphate, 0.01 M KPO,, 0.001 M EDTA, and 0.002 M DTT, clarified by centrifugation at 100 000 g for 1 h, and further purified by column chromatography with Bio-Gel A-l.5 M eluting with the same buffer. Myosin was eluted in the fractions constituting the initial peak. The purity was established by sodium dodecyl sulfate (SDS) gel electrophoresis. The yield of myosin was 2 to 4 mg/g of wet muscle. Sodium dodecyl sulfate polyacrylamide

electrophoresis

SDS polyacrylamide gel electrophoresis according to the technique of Weber

was carried and Osborn

out in 12% acrylamide gels [36]. Myosin solutions, in F2

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T.

C.

SMITHERMAN

ET

AL.

0.04 M Na pyrophosphate, 0.001 M EDTA, were made lo/, with respect to mercaptoethanol and SDS by addition of 107; solutions of these reagents and boiled 3 to 5 minutes before electrophoresis. The gels were stained with Coomassie blue. ATPase

determinations

K+(EDTA)and Ca2+-activated myosin ATPase activities were determined by the pH-Stat method [9] at 30 * O.l”C d in an argon atmosphere. The validity of the technique in our hands was verified by obtaining values that were the same, within experimental error, as those obtained by Pi determination by minor modifications of the Fiske-SubbaRow method [30]. The reaction was started by the addition of 1 ml of 0.04 M ATP and 0.5 M KCl, pH 7.5 to 5 ml of 0.5 M KCl, pH 7.5 solution containing either 0.002 M EDTA [K+(EDTA)-activated] or 0.02 M CaCl, (Ca2+-activated) and 4 ml of a solution containing 0.5 M KC1 and either 0.4 mg [K+(EDTA)-activated] or 2 mg (Ca2+-activated) of myosin. The titrant was 0.0200 M NaOH. The rate of reaction was computed by the equation mEq

Pi mg-l

min-1

=

(ml base added) (Aminutes)

(0.0200

mEq ml-l)

(mg myosin)

(0.83)

where 0.83 is a correction factor for the presence that the pK for phosphate is 6.8. Preparation

of synthetic (reconstituted)

of [H,PO&l

at pH 7.5, assuming

myosin jilaments

Synthetic myosin filament preparations were made from purified myosin by dialysis against buffer containing 0.1 M KCl, 0.01 M KPO,, pH 7.1, conditions known to favor this form [12]. Presence of myosin filament was confirmed by the appearance of turbidity which did not clear with centrifugation at 30 000 g. Dissociation of tight chains from heavy chains during exposure of synthetic myosin jlament to acidic conditions Aliquots of the weakly buffered synthetic myosin filament preparation were transferred to test tubes containing 0.1 M KCl, 0.1 M KPO, at pH 5.0, 5.5, 6.0, 6.5 or 7.0. These samples were gently stirred for 20 min at 22°C and centrifuged at 30 OOOg for 15 min. During some experiments, a fraction of this preparation was returned to pH 7.0 by addition of 0.1 M KOH prior to centrifugation to determine if reassociation of light chains occurred. The supernatant solution was dialyzed against deionized water to precipitate any myosin that remained in solution and centrifuged again. The combined myosin pellets were redissolved in 0.5 M KCl, 0.15 M KPO,, pH 6.8. Both the redissolved pellet and equal aliquots of each supernatant solution were subjected to SDS gel electrophoresis. Densitometry

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scans at 600 nm of these gels were performed in a Gilford 240 Spectrophotometer. Areas under the light chain peaks of these scans were obtained by planimetry. The ATPase activity of the acidified myosin samples was determined immediately after being redissolved and having the pH adjusted to 7.5 by the addition of dilute KOH. Assay for proteolytic enzyme contamination

of purijed

myosin

In order to determine if any enzymes with proteolytic activity under mildly acidic conditions were adsorbed onto the rabbit skeletal or canine cardiac myosins during their preparation, both of these myosin preparations were assayed for caseinolytic activity at pH 6.0. One ml aliquots containing 1 mg of rabbit skeletal or canine cardiac myosin were incubated for 30 min at 37°C at pH 6.0 with 1 ml of a 1 O,&solution of casein yellow (Calbiochem). One ml samples containing only buffer or 0.001 to 0.1 mg of trypsin (from bovine pancreas, Sigma Chemical Co.) were incubated simultaneously. After precipitation of protein with 3 ml of cold 5% trichloracetic acid, the pH of the supernatant solution was adjusted to 12 with about 150 ~1 of saturated NaOH solution and the A,,, was determined.

Solubiliration

of myosin

During the course of these experiments, it was found that myosin was soluble at low pH at low ionic strength, a phenomenon that we have not seen described in the literature. The solubility of myosin at low pH was investigated by gradually lowering the pH of a suspension of myosin in 0.1 M KC1 from pH 7.0 to 3.0 by dropwise addition of 0.5 M H, PO,. The effect of salt concentration on acid-soluble myosin was examined by addition of small amounts of solid KC1 to vary the concentration from 0.1 to 0.5 M.

3. Results Because of the wealth of information about the structure and properties of rabbit skeletal myosin and because of the ease of its preparation in large amounts, the initial experiments were carried out on this protein. Some experiments with canine and human cardiac myosin were also performed.

PuriJcation

of myosins

Utilization of the procedures outlined in this study for purification skeletal myosin yielded preparations that were virtually free of proteins as determined by SDS gel electrophoresis with the gels to detect any such contaminants (Figure 1, Panel A). Heavy chain,

of rabbit white contaminating heavily loaded the three light

154

T. C. SMITHERMAN

ET AL.

chains, and traces of the intermediate bands generally seen with purified myosin were demonstrated. No bands corresponding to troponin or tropomyosin were seen. Generally, no bands corresponding to actin were seen. Rare actin-contamination never exceeded 1 Oh. Canine cardiac myosin was also highly purified but contained 0 to 2% ofactin as well as heavy chain, the two light chains and intermediate bands (Figure 2).

’ 5.0

5.5

6.0

6.5

PH FIGURE 1. SDS gel electrophoresis of rabbit white skeletal myosin. This gel was loaded with about 90 pg of protein. Panel A shows the stained gel of purified myosin. Heavy chain (HC) and the three light-chains (LC,, LC, and LC,) are demonstrated. Panel B shows the electrophoretic pattern of the supernatant solution following acidification, to the given pH values, of synthetic myosin filaments prepared from purified myosin. An equal 25 ~1 aliquot of the supernatant solution of each synthetic myosin filament acidification was loaded onto the gel.

Exposure of synthetic (reconstituted)

myosin jilaments

to acidic conditions

Exposure of rabbit skeletal synthetic myosin filaments to acidic conditions led to a pH dependent dissociation of a protein that had electrophoretic properties on SDS gels that were identical to those of the heaviest light chain of myosin (LC,) (Figure 1, Panel B). As judged by the intensity of this band, dissociation was maximal at pH 6.0, substantially less at pH 5.5, and barely evident at pH 5.0 and 6.5. A small amount of protein that migrated to the same position as the lightest of the three light chains (LC,) was occasionally seen (Figure 1, right side pattern for pH 6.0). In some instances, there was a faint, diffuse band of low molecular weight material (Figure 1, left side pattern for pH 6.0). No LC, was seen in the supernatant solution. In order to confirm that the protein in the supernatant solution was LC, (as

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opposed to a fragment of heavy chain) and to semiquantitate the amount of dissociated LC,, the ratio of LC, to LC, in the myosin remaining after acid exposure was calculated from the areas under the peaks of the densitometry scan of the SDS gels. (Table 1). The decrease in ratio of LC,: LC, in myosin precipitate corresponded to the appearance of LC, in the supernatant solution, and was maximal at pH 6.0. (Table 1). From pH 5.0 to 6.5, there was loss of LC, which was maximal at pH 6.0. However, at all pH values, when the pH was returned to

FIGURE 2. SDS gel electrophoresis of canine cardiac myosin. The gel on the right is from purified myosin. Heavy chain (HC) and the two light chains (LC, and LC,) of myosin are demonstrated. A small amount of actin (A) contamination was demonstrated [faint band approximately midway between heavy chains and the heavier light chain (LC,)]. The amount of contamination by actin was determined by planimetry of the densitometry scan of the stained gels at 600 nm. The amount of actin was usually 0 to 2%. The gel on the left is from the supernatant solution following acidification at pH 6.0 of synthetic myosin filaments from canine cardiac myosin. The center gel is from redissolved myosin following acidification at pH 6.0 in the form of synthetic myosin filament.

156

T. C. SMITHERMAN

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AL.

neutrality at the completion of acid exposure (but before myosin was precipitated and redissolved in 0.5 M KCl) there seemed to be reassociation of LC, to myosin as no LC, could be discerned from the SDS gels of the supernatant solution. Acidification of canine cardiac synthetic myosin filaments at pH 6.0 led to similar dissociation of a protein with electrophoretic properties on SDS gels that were identical to LC, (Figure 2). However, there was also some dissociation of LC,, the lighter of the two light subunits of cardiac myosin. In the example illustrated the weight ratio of LC,: LC, in native myosin was 1.9 (Figure 2, gel on right). In the supernatant solution following acidification at pH 6.0, bands that migrated to the same position as LC, and LC, were demonstrated; the LC,: LC, ratio was 3.0 (Figure 2, gel on left). The LC,: LC, ratio of myosin recovered after acidification was 0.9, consistent with acidification-mediated dissociation of an amount 01’ LC, greater than that of LC, (Figure 2, center gel). As with rabbit skeletal myosin, loss of light chains from the myosin recovered after acidification is confirmatory that the proteins in the supernatant solution are light chains rather than degradation products of heavy chain. The dissociation of some LC, from cardiac myosin prevented determination of the amount of LC, dissociated from acidified synthetic myosin filaments in the manner that was utilized for skeletal synthetic myosin filaments. No caseinolytic activity of either the rabbit skeletal or canine cardiac myosin preparations could be demonstrated at pH 6.0, the value at whichithe dissociation of light chain was maximal. The A4a3 at pH 12 of the supernatant solution remaining after protein precipitation of the incubation of casein yellow with myosin samples was identical to samples containing only buffer. The assay with casein yellow that was utilized was able to detect proteolysis at pH 6.0 with as little as 0.001 mg of trypsin which led to a rise of about 0.010 absorbance units at 423 nm. Acidification of rabbit skeletal and canine and human cardiac synthetic myosin filaments was associated with a pH-dependent decrease in ATPase activity of redissolved myosin from the myosin filaments (Tables 1 & 2). In both canine and human heart synthetic myosin filaments, for which both K+ (EDTA)and Ca2+-activated myosin ATPase were measured, acidification led to approximately equal diminution of monovalent and divalent cation activated ATPase. However, the diminution of myosin ATPase activity following acidification of synthetic myosin filament differed from dissociation of light chains in two important respects. First, the decrease in ATPase activity was progressive with increasing acidification. Second, the loss of enzymatic activity was not reversible when synthetic myosin filaments were returned to pH 7.0 before myosin was precipitated and redissolved. The magnitude of decreased myosin ATPase activity with decreasing pH was similar for acidified skeletal and cardiac synthetic myosin filaments. Solubilization The forms of myosin

present

under

of rabbit white skeletal myosin several conditions

of ionic strength

and pH are

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AL.

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INFARCTION

shown in Table 3. In 0.1 M KCl, with a decrease in the pH from 7.0 to about 6, the material became progressively turbid and between pH 4.5 to 6.0 took the form of a gelatinous aggregate. Below pH 4.4 myosin became progressively soluble and below pH 4.0, the solubility exceeded 6 mg/ml. With virtually no salt (only sufficient H,PO, or HCl to lower the pH) myosin was similarly soluble below pH 4. At pH 3.0 to 3.5, myosin became insoluble at salt concentrations above 0.15 M KCl. As is widely recognized, myosin exists as a precipitated thread-like aggregate in 0.3 M KC1 at the isoelectric point of 5.5 [12].

TABLE

3. Myosin

forms

at different

pH Ionic

do.1

values

and

2 0.3

Form

6.1-7.0

Myosin

4.5-6.0

Gelatinous

3.0-4.4

Soluble

filament aggregate

strengths

strength

M KC1

PH

ionic

M

KC1 Form

PH 6.0-7.0

Soluble

5.5-6.0

Thread-like

3.0-5.4

Denser,

aggregate thread-like

aggregate

4. Discussion The results of this study indicate that when synthetic myosin filaments prepared from purified myosin are acidified with dilute buffers, there is partial dissociation of myosin light chains from heavy chains. In rabbit skeletal myosin, this dissociation was limited to the two light chains, LC, and LC,, which are also dissociated by exposure to pH values above 10.5 and have been called the “alkali light chains” [37]. With canine cardiac myosin, in addition to LC,, a smaller quantity of LC, is also dissociated. Alkali dissociation of cardiac myosin is also known to dissociate LC, as well as LC, [22]. Proteolysis of rat cardiac myosin, probably owing to contamination of the contractile protein with an endogenous protease, has recently been described [3.5]. However, it seems unlikely that the results reported in the present study are due to contamination of the myosin preparations by proteolytic enzymes. No caseinolytic activity of these myosin preparations could be detected at pH 6.0, at which the dissociation of light chains was most marked. The possibility is remote that the proteins that are removed from acidific synthetic myosin filaments are proteolytic degradation products of myosin heavy chains that fortuitously co-migrate with myosin light chains during SDS gel electrophoresis, since there was a decrease in light chain content of myosin after acidification of reconstituted myosin filaments that corresponded to the appearance of free light chains in the acid buffer.

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T. C. SMITHERMAN

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AL.

Furthermore, the protease contamination of cardiac myosin noted from rat heart preparations was not noted during myosin purification from dog heart [3.5]. Acid dissociation of light chains from synthetic myosin filaments occurred from pH 5.0 to 6.5 and was maximal at pH 6.0. Intracellular pH values of ischemic heart muscle obtained by several techniques have been reported to be in the range of 5.7 to 6.6 compared with the normal intracellular myocyte pH of about 7.0 [S, 11, 19, 261. While none of the currently available techniques for measuring intracellular pH is ideal [reviewed in 241, there is wide-spread agreement that this acidosis occurs and is likely to play an important role in the early pump failure of the ischemic heart and may be important in contributing to irreversible damage to the myocytes. The intracellular proton concentration in the ischemic myocyte appears to be near that observed for maximal dissociation of light chains from synthetic myosin filament id vitro. Whether this dissociation would be mitigated or prevented by the presence of the other contractile proteins in vivo is unknown. However, free light chains in the cytosol would presumably cross the damaged sarcolemma following infarction as do other intracellular proteins, many of which are used as diagnostic clinical tests for myocardial necrosis in patients with suspected myocardial infarction. Indeed, such appearance of myosin light chains in the peripheral circulation after myocardial infarction has recently been reported in men [33] and following experimental infarction in dogs [18]. It has been demonstrated that myosin light chains and heavy chains are synthesized independently [2] and that a small amount of light chains not bound to heavy chains are present in the myocyte [IO]. However, there is an insufficient quantity of unbound light chains to account for the amount of circulating light chains that have been reported following myocardial infarction [7]. Acid-mediated dissociation of myosin light chains from myosin filament such as that found in the present study could account for these reported amounts of circulating myosin light chains and lends credence to the idea that the results reported here in vitro may be a reflection of a similar phenomenon in vivo. The results of the present study also demonstrate a decrease in the enzymatic activity of myosin when synthetic myosin filaments prepared from purified myosin are slightly acidified with dilute buffers. Since the light chains of myosin are known to be essential for its enzymatic activity [8, 291 dissociation of light chains such as demonstrated in the present work would be expected to lead to a decrease in ATPase activity. While a pH dependent decrease in ATPase activity was found in our studies, it does not appear possible to attribute this change exclusively to dissociation of light chains for three reasons: (1) the diminution in ATPase activity in the lower pH range was substantially greater in magnitude than was dissociation of light chains, (2) the diminution of ATPase activity progressed with decreasing pH while dissociation of light chains was maximal at pH 6.0, and (3) on return of the pH to neutrality prior to precipitating and redissolving myosin, there was reassociation of light chains to heavy chains without

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recovery of ATPase activity. However, it is possible that reassociation of light chains to heavy chains under the conditions of these experiments occurred in a non-specific fashion that did not restore protein structure to that necessary for enzymatic activity. While there is some evidence that periods of ischemia or hypoxia long enough to impair cardiac contractility do not cause irreversible damage to the contractile proteins [I, 3, 4, 5, 131 it is conceivable that acute ischemia-induced alterations to the contractile proteins could occur in vim, but be reversed during the process of extraction and purification of the preparations that were studied. As little as 30 min of acute anoxia was reported to cause irreversible damage to cardiac myosin as manifested by diminished ATPase activity [21]. Furthermore, a recent investigation of the ATPase activity of myosins isolated from myocardium at various times after experimental infarction in dogs reported an initial increase in ATPase activity (12 to 24 h after infarct) followed by a marked decrease (48 h to 14 days after infarct) [14]. The possibility that the intracellular adicosis of acute ischemia may depress actomyosin ATPase activity has received some attention in recent years. A decrease in actomyosin ATPase activity with decreasing pH was found with in vitro experiments with both skeletal [17, 2.5, 281 and cardiac [17, 381 myofibrils. The mechanism for this decreased ATPase activity is unclear. Katz & Hecht [15] proposed that the precipitous decline in cardiac contractility observed after coronary artery ligation could be due to depressed actomyosin activity resulting from an acidosis-induced decrease in Ca +2 binding to troponin. This has been investigated in several laboratories recently with conflicting results [reviewed in 311. A second possible mechanism, that increased hydrogen ion concentration interferes with ATP binding to myosin, appears to be unlikely, in light of work by Williams et al. [38]. A third possibility [38] that increased hydrogen ion concentration decreases the rate of ATP hydrolysis at each myosin-actin cross bridge, has not been investigated to our knowledge. The results of the present study bear on a fourth possibility [38] that increased hydrogen ion concentration interferes with the myosin ATPase of the myofibril irrespective of the effect on the troponin tropomyosin regulatory complex. While extrapolation of these results with reconstituted myosin filaments in vitro to the myofilament of the intact heart must be made with caution, the loss of myosin ATPase activity following acidification of synthetic myosin filaments provides a possible mechanism to explain at least part of the decreased ATPase activity of acidified myofibrillar preparations. Acknowledgements We acknowledge with gratitude Melba Loveall for their technical assistance.

Messrs. Paul Jordan, Robert Butsch and Miss assistance, and Mrs Betty Bibus for her clerical

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21. 22.

23.

24. 25.

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27. 28.

29. 30.

31.

32. 33.

34. 35.

36.

37. 38.

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