Biochimica et B:opl~vsica Acta. 1074(1991) 95-100 © 1991 ElsevierScience PublishersB.V. 0304-4165/91/$03.50 ADONIS 0304416591001593
Alterations in cardiac contractile proteins due to oxygen free radicals S h i n g o S u z u k i , M a s a n o r i K a n e k o , D o n a l d C. C h a p m a n
a n d N a r a n j a n S. D h a l l a
Division of Cardiot'ascular Sciences, St. Boniface General Hospital Research Centre a,,d ~'partment of Pl~'csivh gv. Faculty of Medicine, Unit'ersity of Mmlitoba. Wimupeg {Canada)
(Received 29 October 1990) Key words: ATPase.Mg2+-:ATPase,Ca2 +-stimulated:Sulfhydrylgroup: Superoxideanion; Hydrogenperoxide: Hypochlorousacid; (Rat heart myofibril) In view of the potential role of free radicals in the genesis of cardiac abnormalities under different pathophysiological conditions and the importance of contractile proteins in determining heart function, this study was undertaken to examine the effects of oxygen free radicals on the rat heart myofibrils. Xanthine plus xanthine oxidase (X + XO) which is known to generate superoxide anions ( O z ) and hydrogen peroxide (H20~), an activated species of oxygen, was found to decrease CaZ+-stimulated ATPase activity, increase MgZ+-ATPase activity and reduce sulfhydryl (SH) group contents in myofihrils; these effects were completely prevented by superoxide dismutase (SOD) plus catalase (CAT). Both HzO 2 and hypochlorous acid (HOCi), an oxidant, produced actions on cardiac myofibrils similar to those observed by X + XO. The effects of H 2 0 z and HOCI were prevented by CAT and L-methionine, respectively. N-ethylmaleimide (NEM) and 5,5'-dithiobis(2-nitrobenzoie acid) (DTNB), inhibitors of S H groups, also produced effects similar to those seen with X + XO. Dithlothreitul (DTr), a well known sulfhydryl-redueing agent, prevented the actions of X + XO, H2Oz, HOCI, NEM and DTNB. These results suggest that marked changes in myofibrillar ATPase activities by different species of oxygen free radicals may he mediated by the oxidation of S H groups.
Introduction
Oxygen free radicals have been suggested to participate in the pathophysiology of contractile failure and cellular damage due to ischemia-reperfusion injury in the heart [1-3]. Furthermore, different oxygen free radical generating systems have been shown to induce contractile failure, muscle contracture and cell damage in the myocardium [4-6]. Since the function of the heart is primarily determined by the interaction of Ca 2 + with contractile proteins as well as by the regulatory activities of sareolemma and sarcoplasmic reticulum [7,8], it is likely that adverse effects of oxygen free radicals in the heart are due to their actions on the functions of one or more of these organelles. Earlier studies in this
Abbreviations: X, xanthine; XO, xanthine oxidase; SH, sulfhydryl group; SOD, superoxidedismutase; CAT, catalase: NEM, N-ethylmaleimide: DTNB. 5,5'-dithiobis(2-nitrobenzoicacid). Correspondence: N.S. Dhalla, Division of Cardiovascular Sciences, St. BonifaceGeneral Hospital Research Centre, 351 Tache Avenue, Winnipeg,Manitoba. Canada R2H 2A6.
regard have revealed that Ca2+-transport activities of both cardiac sarcolemma [9-12] and sarcoplasmic reticulum [13,14] are depressed upon treating these membranes with oxygen radical generating systems. However, virtually nothing is known at present concerning the effects of oxygen free radicals on myofibrils, the basic contractile machinery of the myocardial cell. This study was therefore undertaken to investigate the actions of oxygen free radical generating systems on myofibrillar ATPase activities. Since sulfhydryl groups are required for the myofibrillar ATPase activities [1517] and oxygen radicals are known to oxidize sulfhydryl groups in proteins [10,18-20], it is possible that the free radical-induced changes in myofibrillar ATPase activities are associated with alterations in myofibriUar sulfhydryl group contents. Accordingly, sulfhydryl group contents were measured upon treatment of myofibrils with oxygen free radical generating systems in order to provide some information regarding the mechanism of changes observed in this study. The effects of different free radical scavengers on alterations in myofibrillar ATPase and sulfhydryl groups were also examined to verify if the observed changes are mediated through free radicals.
Materials and Methods
Materials" Xanthine. superoxide dismutase (3600 units/mg), catalase (14100 units/mg), L-methionine, DTT, ATP, DTNB, and NEM were purchased from Sigma. Xanthine oxidase (12.45 units/ml) was the product of Calbiochem. Sodium hypochlorite (NaOCI), hydrogen peroxide, and sodium dodecyl sulfate (SDS) were from Mallinckrodt, Fisher Scientific. and Bio-Rad, respectively. All chemicals used were of analytical grade.
Methods Preparation of myofibrils. Male Sprague-Dawley rats weighing approx. 200 g were used. Animals were killed by decapitation, hearts were removed, atria and any large vessels were carefully trimmed, and the ventricular tissue was processed for the isolation of myofibrils by the method of Solaro et al. [21]. The myofibrillar fraction was suspended in a final solution containing 100 mM KCI, 20 mM Tris-HCl (pH 7.0). The protein concentration was determined by the method of Lowry et al. [22] using the bovine serum albumin as a standard. Measurement of Mg2 +-A TPase and Ca2 +-stimulated A TPase activities" of myofibrils. The experimental conditions were the same as reported elsewhere [23]. Briefly, for the estimation of Mg~+-ATPase activity, myofibrils (0.5 mg protein/ml) were preincubated at 30 o C for 5 min in 1.0 ml of medium containing: 25 m M imidazole (pH 7,0), 2 mM MgCI z, 5 mM NAN3,1 mM EGTA and 50 mM KCI. Appropriate blank tubes contained all of the above with the exception of myofibrillar proteins. The reaction was started by the addition of 2 m M ATP and terminated 5 min after by the addition of 1 ml of 127o trichloroacetic acid. These samples were centrifuged and phosphate in the protein-free supernatant was determined by the method of Taussky and Shorr [24]. Total (Ca 2÷ plus Mg 2+) ATPase activity was determined in the medium containing: 25 m M im. idazole (pH 7.0), 2 mM MgC!z, 5 m M NAN3, 50 m M KCI and 0.01 m M free Ca 2÷. The concentration of free Ca 2÷ in the medium was adjusted by using EGTA as previously described [23]. Ca2+-stimulated ATPase activity was taken as the difference between the values obtained for total and Mg2+-ATPase activities. Determination of sulfhydryl content in myofibrils. Total sulfhydryl groups in myofibrils were determined with DTNB [25]. Myofibrils (0.2 mg protein/ml) were incubated in 60 mM imidazole (pH 7.4) containing 30 mM EDTA and 0.8% SDS in a final volume of 2.58 ml. The reaction was started by adding DTNB at a final concentration of 2 mM. Absorbance was measured at 412 nm 5 min after but this value did not change within 30 min of incubation. The reference cuvette contained solvents and DTNB concentrations identical to the samples, but myofibrillar proteins were omitted. After re-
cording the final absorbance, 5 mg NEM was then added to the sample and reference cuvettes; the value obtained 5 min later was subtracted from the value in the presence of DTNB. The concentration of sulfhydryl groups was calculated by using an E molar value of 13 600 [25]. Free radical-generating systems and oxidants. Superoxide anion radicals were generated by xanthine oxidase (0.03 U / m l ) reaction by using xanthine (2 mM) as a substrate. In this experiment xanthlne oxidase was dialyzed overnight against 0.01 M Tris-HCI buffer (pH 7.4) at 4 ° C in order to eliminate the contamination by (NH4)2SO4 and EDTA which are included in the commercial preparation. Subsequently, it was pretreated with 0.4 m M phenylmethylsulfonyl fluoride (PMSF) to inhibit the associated trypsin-like activity. Hydrogen peroxide was used at the concentration of 1 raM. HOCi, a well known oxidant, was prepared by vacuum distillation of NaOCI after adjusting the pH to 6.2 with dilute sulfuric acid. The concentration of HOCI was determined using a molar absorptivity coefficient of 100 at 235 n m [26,27] and was used at the concentration of 0.4 m M in this experiment. SOD (80 # g / m l ) , CAT (10 /tg/mi), and L-methionine (1 mM) were used as scavengers for 02-, H 2 0 ~, and HOCI, respectively [9,28,29]. D T T (1 mM) was also used t, obtain a protection from the effects of these activated oxygen species [10]. The combination of H202 (0.1 mM) and Fe z+ (0.1 raM) was used to generate hydroxyl radicals [9,30]. It should be pointed out that different species of radicals are formed by the oxygen radical generating systems employed in this study and different scavengers are also non specific in nature. It is therefore not our intention to emphasize the specificity of different radical generating systems or the scavengers used here. Statistical analysis. For the determination of ATPase activity and sulfhydryl group content, all assays were carried out in duplicate by using different preparations. Results were expressed as means + S.E. For statistical evaluation, Student's t test was used. P level < 0.05 was taken to reflect a significant difference between control and treated preparations. Results
Effects of xanthine plus xanthine oxidase on heart myofibrillar A TPase activities and SH group contents The effects of 2 m M X plus 0.03 U / m l XO, a predominantly superoxide anion generating system in which some H202 is also produced, on heart myofibrillar ATPase activities were examined. Fig. 1 shows the time-dependent effects on Ca 2 +-stimulated ATPase and MgZ+-ATPase activities. After 10 min incubation of mvofibrils with X plus XO, a significant difference was apparent in Mg2+-ATPase activity but Ca2+-stimulated ATPase activity was not affected. While MgZ+-ATPase
97
-~'12 ~
0-0
Co2+
"~
0-0
Mg 2 +
TABLE I
- 6
Effects of X + X O on myofibrillar ATPase activities and S H group contents
,
~10 ~--& , ~
T , 0-_____._.__.__6
•
:~ -~
~
38
.E_ +1 •
,
,
10 20 30 60 Incubotion Time (min) Fig, I. Incubation time-dependent effects of xanthine plus xanthine oxidase on heart myofibrillar ATPase activities. Myofibrils (5 mg protein/ml) were preincubated at 30°C with xanthine (2 mM) plus xanthine oxidase (0.03 U/ml) in a medium containing 50 mM KCI, l0 mM Tfis-HCI (pH %0) in a total volume I ml, and then ATPase activities were assessed. Each value is a mean±g.E, of six preparations. • P < 0.05 vs. control.
Each value is a mean+S.E, of six to nine different preparations. Myofibrils (5 mg protein/ml) were preincubated for 20 rain at 30°C in a medium containing 50 mM KCI, 10 mM Tris-HCI (pH 7.0) plus the addition, shown in a total volume I ml. Aliquots of 0.l ml samples were assayed for ATPase activities and SH group contents. Final concentrations of xanthine (XL xanthine oxidase (XOL superoxide dismutase (SOD), and catalase (CAT) were 2 mM, 0.03 U/ml, 80 tLg/ml, and tO ~g/ml, respectively. * P < 0.05 vs, control and * * P < 0.05 vs. X + XO+ SOD.
X+XO X+XO+SOD X+XO+SOD +CAT X XO SOD CAT
(~mol/mg protein/h) ~ stimulated
contents (nmol/mg protein)
4.97 + 0.16 * 5.13+0.27"
6.48:i: 0.18 * 7.60-+@29*
54.2+1.0" 59,1 ± l.l*
10.30+0.30"* 10.34+0.31 9.89 + 0.28 10.40 + 0.25 10.34_+0.41
69.5-+0.8 "* 67.2_+1.8 68.5 + 2.2 65.5 + 1.9 66.6+2.9
2.60+0.17"* 2.e0+0.23 2.88 =i=0.23 2.67 + 0.08 2.92_+0.17
0
activity d i d n o t increase further, C a 2 + - s t i m u l a t e d A T P a s e activity w a s progressively i n h i b i t e d d u r i n g a 20 to 60 rain i n c u b a t i o n period. M y o f i b r i l s i n c u b a t e d w i t h o u t X o r X O for the s a m e p e r i o d s d i d n o t s h o w a n y c h a n g e in M g 2 + - A T P a s e o r C a 2 + - s t i m u l a t e d A T P a s e activities ( d a t a n o t shown). T a b l e 1 s h o w s t h a t i n c u b a t i o n o f m y o f i b r i l s with either 2 m M X, 0.03 U / m l X O , 80 ~ t g / m i S O D , o r 10 p , g / m l C A T for 20 m i n at 3 0 ° C d i d not a f f e c t the A T P a s e activities. A c o m b i n a t i o n o f X plus X O d e p r e s s e d C a 2 + - s t i m u l a t e d A T P a s e activity a n d increased M g 2 + - A T P a s e activity b y 37 a n d 96% o f the c o n t r o l values, respectively. S O D s h o w e d a p r o t e c tive effect o n the d e p r e s s i o n in C a 2 + - s t i m u l a t e d A T P a s e activity b u t there w a s n o effect o n the increase in M g 2 + - A T P a s e activity. T h e c o m b i n a t i o n o f C A T w i t h S O D w a s m o r e effective t h a n S O D a l o n e in p r e v e n t i n g c h a n g e s d u e to x a n t h i n e plus x a n t h i n e oxidase. It m a y b e n o t e d t h a t c o n t r o l values for m y o f i b r i l l a r M g 2+A T P a s e a n d C a = + - s t i m u l a t e d A T P a s e activities a r e similar to those r e p o r t e d elsewhere [17,23]. T h e d a t a in T a b l e I also s h o w s t h a t S H g r o u p c o n tents were d e c r e a s e d b y X plus X O ; S O D s h o w e d s o m e p r o t e c t i v e effect o n S H g r o u p c o n t e n t s . T h e c o m b i n a -
tion o f c a t a l a s e w i t h S O D i n d i c a t e d c o m p l e t e p r o t e c tion f r o m the d e p r e s s i o n o f S H g r o u p c o n t e n t s inc u b a t e d with X p l u s X O . T h e i n c u b a t i o n of m y o f i b r i l s w i t h e i t h e r 2 m M X, 0.03 U / m l X O , 8 0 / ~ g / m i S O D , or 10 p , g / m i C A T for 20 rain at 3 0 ° C d i d n o t affect the S H g r o u p c o n t e n t s . In o r d e r to test the role o f c h a n g e s in S H g r o u p c o n t e n t s in X p l u s X O i n d u c e d a l t e r a t i o n s in m y o f i b r i l l a r A T P a s e activities, the effects o f different c o n c e n t r a t i o n s o f D T T w e r e e x a m i n e d . T h e results in Fig. 2 i n d i c a t e t h a t a significant p r o t e c t i v e a c t i o n of D T T o n the d e p r e s s a n t effect o f X p l u s X O for C a -'+-
r c2
4~
!li!ii!!H
0 on rol
100 1000 0 Concentrationof DTT iuM) Fig. 2. Dose-dependent effects of dithiothreitol (DTT) on myofibrillar ATPase activities in the absence (control) or presence of xanthine (X) plus xanthin¢ oxidase (XO). Myofibrils (5 mg protein/nil) were preincubated for 20 rain at 30* C with various concentrations of DTT with or without 2 mM X plus 0.03 U/ml XO in a medium containing 50 mM KCI and 10 mM Tris-HCI (pH 7.0). Each value is a mean :t: S.E. of four different preparations. * P < 0.05 vs. 0 iX plus XO without DTr).
s t i m u l a t e d A T P a s e activity w a s evident at 10 # M c o n c e n t r a t i o n ; m a x i m a l a c t i o n w a s seen at 50 p M c o n c e n t r a t i o n o f D T T . O n the o t h e r h a n d , X + X O i n d u c e d increase in M g 2 + - A T P a s e w a s significar~tly p r o t e c t e d at 50 ttM c o n c e n t r a t i o n ; c o m p l e t e p r o t e c t i o n w a s seen a t 100 # M D T T .
Effects of hydrogen peroxide and hypochlorous acid on A TPase actioities and S H group contents T h e results in T a b l e II i n d i c a t e t h a t m y o f i b r i l l a r C a 2 + - s t i m u l a t e d A T P a s e activity a n d S H g r o u p c o n tents were decreased w h e r e a s M g 2 + - A T P a s e activity w a s increased b y 1 m M H 2 0 2. T h e s e c h a n g e s in myofibrils were p r o t e c t e d b y 10 t t g / r n l C A T . T h e inhibition o f C a 2 + - s t i m u l a t e d A T P a s e activity a n d the s t i m u l a t i o n of M g 2 + - A T P a s e activity b y H 2 0 2 w e r e
~.}'~[ "e~+-~,,mo,ot,,,, 0 M ~ "+
16
Control
DTN8
il
DTNB NEM NEId DTT DTT Fig. 3. Effects of SH reagents on myofibrillar ATPase activities. Myofibrils (5 mg protein/ml) were preincubated for 20 min at 30°C with 0.1 mM DTNB or 0.05 mM NEM in the absence or presence of 1 mM DTT in a medium containing 50 mM KCI and 10 mM Tris-HCI (pH 7.0). Values are means:l:S.E, of five to eight different preparations. * P < 0.05 vs. control and *P < 0.05 vs. DTNB or NEM.
TABLE 11
Effects of H20, on myoflbrillarATPase activities and S H group contents Each value is a mean+S.E, of four to five different preparations. Myofibrils (5 mg protein/ml) were preineubated for l0 rain at 30 o C in a medium containing 50 mM KCI, l0 mM Tds-HCI (pH 7.0) in the absence or presence of different interventions indicated below. Final concentrations of H202, catalase, and DTT were 1 raM, 10 #g/ml, and 1 raM, respectively. * P < 0.05 vs. control and * * P < 0.05 vs. H 202 .
Control HzOz H202 +CAT H202 + DTT
ATPase activity (ttmol/mg protein/h) Mg2+ Ca 2+. stimulated
SH group contents (nmol/mg protein)
2.96 + 0.l 2 5.464-0.18" 2.924-0.16"* 2.684-0.13"*
66.3 :l: 2.7 47.6:1:2.6" 65.84-2.3**
10.89:1:0.45 5.92:1:0.38" 10.404-0.53"* 10.194-0.41"*
TABLE II1
Effects of HOCI on myofibrillar ATPase activities and SH group contents Each value is a mean+S.E, of five to six different preparations. Myofibrils (5 mg protein/ml) were preincubated for 10 rain at 30°C with or without various additions in a medium containing 50 mM KCI, 10 mM Tris-HCI (pH 7.0). Final concentrations of hypochlorous acid (HOCI), L-methionine (Meth), and DTT were 0.4 raM, l raM, and 1 mM, respectively. * P < 0.05 vs. control and ** P < 0.05 vs. HOCI.
Control HOCI HOCI+ Meth HOCI+ DTI" Meth DTT
ATPase activity (/~mol/mg protein/h) Mg2 + Ca2+. stimulated
SH group contents (nmol/mg protein)
2.80+0.12 9.51 +0.16 * 3.79+0.16"* 3.55+0.14"* 2.80+0.11 2.80 :l:0.13
67.6+1.1 35.7+ 2.9 * 59.84-0.9** 67.24-0.4
10.96+0.15 5.73:1:0.31* 11.644-0.12"* 11.09+0.19"* 11.20+0.16 11.034-0.19
s i g n i f i c a n t l y b l o c k e d b y 1 m M D T T . T o assess the effects o f H O C I o n A T P a s e activities a n d S H g r o u p c o n t e n t s , m y o f i b r i l s w e r e i n c u b a t e d for 10 r a i n at 30 o C with this o x i d i z i n g a g e n t ( T a b l e III). H O C 1 (0.4 m M ) d e p r e s s e d b o t h C a 2 + - s t i m u l a t e d A T P a s e activity a n d S H g r o u p c o n t e n t s b u t i n c r e a s e d the M g 2 + - A T P a s e activity. T h e s e c h a n g e s w e r e s i g n i f i c a n t l y p r e v e n t e d b y 1 m M L - m e t h i o n i n e . H O C I i n d u c e d c h a n g e s in M g 2+A T P a s e a n d C a 2 + - s t i m u l a t e d A T P a s e w e r e also p r e v e n t e d b y 1 m M D T T . It c a n b e seen f r o m T a b l e III that L-methionine alone did not show any significant effects o n A T P a s e activities o r S H g r o u p c o n t e n t s . Likewise, D T T a l o n e d i d n o t a f f e c t the m y o f i b r i l l a r A T P a s c a,;fivities. It s h o u l d also b e m e n t i o n e d t h a t e x p e r i m e n t s to test the effects o f h y d r o x y l radicals, w h i c h a r e p r o d u c e d b y F e 2+ p l u s l o w c o n c e n t r a t i o n s o f H 2 0 2 , c o u l d n o t b e t r i e d b e c a u s e 0.1 m M F e 2+ h a s a m a r k e d a c t i o n in i n c r e a s i n g a n d d e p r e s s i n g the m y o f i b r i l l a r M g 2 + - A T P a s e a n d Ca2 +-stimulated A T P a s e activities, respectively ( d a t a n o t shown).
Effects ojo~ ffhydryl reagents on M g 2 +- and C a : +-stimulated A T r , ~ e activities T o e x a m i n e the role o f S H g r o u p s in a l t e r i n g the m y o f i b r i l l a r A T P a s e activities, the effects o f D T N B a n d N E M , well k n o w n i n h i b i t o r s o f S H g r o u p s , w e r e inv e s t i g a t e d u n d e r the e x p e r i m e n t a l c o n d i t i o n s e m p l o y e d in this s t u d y (Fig. 3). C a 2 + - s t i m u l a t e d A T P a s e activity w a s depres,,~d w h e r e a s M g Z + . A T P a s e activity w a s increased b y 0.1 m M D T N B o r 0.05 m M N E M . D T T (1 m M ) s h o w e d p r o t e c t i v e effects o n the c h a n g e s in m y o f i b r i l s d u e to D T N B o r N E M . Discussion In this s t u d y w e h a v e s h o w n t h a t t r e a t m e n t o f h e a r t m y o f i b r i l s w i t h X p l u s X O a n d H2Oz d e c r e a s e d the
99 Ca2+-stimulated ATPase activity and incre:~sed the Mg2÷-ATPase activity. Since the effects of X pius XO were completely prevented by a combination of both SOD and CAT and those of H202 were completely prevented by CAT, it is evident that these actions of X plus XO were mediated by the generation of both superoxide anion radicals and H202 in the incubation medium. The observed decrease in Ca2+-stimulated ATPase activity l.~y X plus XO does not seem to be due to corresponding increase in Mg'-+-ATPase activity because (i) a significant increase in Mg 2 +-ATPase activity was not associated with a significant decrease in Ca -'+stimulated ATPase activity upon incubating myofibrils with X plus XO for a period of 10 min, (ii) a marked depression in Ca2+-stimulated ATPase was not associated with a corresponding increase in Mg2+-ATPase activity during 20 to 60 min incubation period of myofibrils with X plus XO, (iii) a significant protection of X plus XO induced decrease in Ca'-+-stimulated ATPase activity by SOD was not associated with a significant protection of X plus XO induced increase in Mg2+-ATPase activity, and (iv) a significant prevention of X plus XO induced decrease in CaZ+-sdmulated ATPase activity was seen at 10 ~tM concentration of D T T whereas 50 # M concentration of DTT was required to demonstrate a significant effect on the changes in Mg'-+-ATPase. In view of the fact that Ca "-+ sensitivity (Ca2+-stimulated ATPase) and basal activity (Mg2+-ATPase) of the myofibriUar ATPase system are manifested due to the interaction of several components such as aetin, myosin, troponin and tropomyosin [15], it is possible that the observed effects of superoxide anions and H20'- are due to their actions on one or more contractile and regulatory proteins. Extensive studies in this regard are required to establish the exact site of action of the oxy radicals on myofibrils. HOCI, which is gererated as an oxidant upon activation of neutrophils [2' ], was found to increase Mg 2+ATPase and decrease the Ca2+-stimulated ATPase activities in rat heart myofibrils. Other oxidants, such as diamide, have also been shown to exert similar effects on the guinea pig heart myofibrillar preparations [32]. The effects of HOCl on myofibrils were prevented by L-methionine, a well known scavenger of this oxidizing agent [31]. Since the actions of HOCI and diamide on myofibrillar ATPases were similar to those observed with superoxide anions and H'-O'-, it is likely that the effects of free radicals are mediated due to oxidative stress on myofibrillar proteins. Although oxygen free radicals and HOCI have also been reported to inhibit several membrane bound enzymes such as sarcoplasmic reticnlar CaZ+-stimulated ATPase as well as sarcolemmal N a + / K ÷ - A T P a s e and Ca'-÷-stimulated ATPase [9,10,12,20,31,33,34], these studies do not provide any evidence as to whether the inhibitory effects on enzyme activities are due to peroxidation of the membrane
lipids or direct oxidation of the membrane proteins. Since the myofibrillar preparation employed in our experiments is devoid of any associated lipids [21], it is reasonable to conclude that the observed effects of free radicals and HOCI are due to direct oxidation of myofibrillar proteins. The experiments reported in this study reveal that changes in myofibrillar ATPase due to superoxide anions, H202 and HOCI were associated with reduction in myofibrillar SH group contents. Furthermore, respective scavengers of these free radicals and oxidants were able to prevent these alterations in myofibrillar SH group contents. In view of a crucial role of SH groups in the expression of myofibrillar ATPase activities [1517], it is possible that the observed effects of free radicals and oxidants on myofibrillar ATPases are due to oxidation of SH groups. This view is supported by the fact that DTT, a well known sulfhydryl reducing agent, was able to prevent changes in myofibrillar ATPases due to X plus XO, H202 and HOCL Furthermore, both DTNB and NEM, well known inhibitors of SH groups [10,17], were found to increase Mg2 +-ATPase and decrease Ca2+-stimulated ATPase activities in myofibrils. It should also be noted that the actions of both DTNB and N E M like those seen with radicals and oxidants were prevented by DTT. It is therefore suggested that the observed effects of oxygen free radicals, H202 and HOCI on myofibrillar ATPase activities are mediated by the oxidation of myofibrillar SH groups, Since oxygen radical species have also been shown to affect amino acids, protein-protein erosslinking, and protein strand scission [18,19], it is not our intention to rule out the actions of free radicals on these sites. Nonetheless, the observed effects of X plus XO, H202 and HOCI on myofibrillar ATPase can be seen to explain the contractile dysfunction of myocardium by these interventions [4-6,35]. The involvement of myofibrillar defects due to the action of free radicals and oxidants in altering the contractile function may not be specific in nature because these interventions have also been reported to affect sarcolemmai and sareoplasmic reticular functions under similar experimental conditions [9-14]. Acknowledgments The work reported in this study was supported b9 a granz from the Medical Research Council of Canada. Dr. S. Suzuki was a postdoctoral fellow of the Medical Faculty at the University of Manitoba during the tenure of this study. References 1 Ambro~io.G. Pecker. L.C- Hulchins. G.M.. Welsman.H.F. and Weisfeldt. M.L. (1986) Circulation 74.1424-1433.
100 2 Jolly, S.R., Kane, W.J., Bailie. M.B.. Abrams. G.D. a~d Lucchesi, 5.R, (1984) Circ. Res. 54, 277-285. 3 epic, L.H. (1989) Circulation 80, 1049-1662. 4 Gupta, M. and Singal. P.K. (1989) Can. J. Physiol. Pharmaeol. 67, 1549-1559. 5 Hammond, B. and Hess, M.L.'(1985) J. Am. Coll. Cardiol. 6, 215-220. 6 Kaminishi. K., Yanagishita, T. and Kako, K.J. (1989) Can. J. Cardiol. 5,168-174. 7 Dhalla, N.S., Ziegelhoffer, A. and Harrow, J.A.C. (1977) Can. J. Physiol. Pharmacol. 55,1211-1234. 8 Dhalla, N.S.. Pierce, G.N., Panagia, V., Singal, P.K. and Beamish, R.E. (1982) Basic Res. Cardiol. 77,117-139. 9 Kaneko, M., Beamish, R.E. and Dhalla, N.S. (1989) Am, J. Physiol. 256. H368~H374. 10 Kaneko, M., Elimban, V. and Dhalla, N.S. (1989) Am. J. Physiol. 257, H804-H81 I. 11 Kaneko, M.. Ice, S.L., V~oil, C.M. and Dhalla, N.S. (1989) J. Mol. Cell. Cardiol. 21,935-943. 12 Xie, Z., Wang, Y., Askari, A., Huang, W.-H., Klaunig, J.E. and Askari, A. (1990) J. Mol. Cell. Cardiol. 22, 911-920. 13 Rowe. G.T., Manson, N.H., Caplan, M. and Hess, M.L. (1983) Circ. Res. 53, 584-591. 14 Okabe, E., Hess, M.L., Oyama, M. and Ito, H. (1983) Arch. Biochem. Biophys. 225, 164-177. 15 Scheuer, J. and Bhan. A.K. (1979) Circ. Res. 45,1-12. 16 Sekine, T., Barnett, L.M. and Kielley, W.W. (1962) J. Biol. Chem. 237, 2769-2772. 17 Pierce, G.N. and Dhalla, N.S. (1985) Am. J. Physiol. 248, E170E175. 18 Freeman, B.A. and Crapo, J.D. (1982) Lab. Invest. 47, 412-426.
19 Nagy, I.Z. and Floyd, R.A. (1984) Biochim. Biophys. Acta 790, 238-250. 20 Seherer. N.M. and Deamer. D.W. (1986) Arch. Biochem. Biophys. 246, 589-601. 21 Solaro, R.J., Pang, D.C. and Briggs, F.N. (1971) Biochim. Biophys. Acta 245. 259-262. 22 Lowry, O.H., Rosebrough. N.J.. Fan'. A.L. and Randall. R.J. (1951) J. Biol. Chem. 193, 265-275. 23 Pierce, G.N. and Dhalla, N.S. (1981) J. Mol. Cell. Cardiol. 13. 1063-1069. 24 Tat, ssky. H.H. and Shorr, E. (1953) J. Biol. Chem. 202, 675-685. 25 Boyne, A.F. and EIIman, G.L. (1972) Anal. Biochem. 46, 639-653. 26 Green, T.R., Fellman. J.H. and Eieher, A.L. (1985) FEBS Lett. 192, 33-36. 27 Morris. J.C. (1966) J. Phys. Chem. 70, 3798-3805. 28 Reeves, J.P., Bailey, C.A. and Hale, C.C. (1986) J. Biol. Chem. 261, 4948-4955. 29 Weiss, S.J.. Lampert, M.B. and Test, S.T. (1983) Science 222. 625-628. 30 Tien, M., Svingen, B.A. and Aust, S.D. (1982) Arch. Biochem. Biophys. 216,142-151. 31 Kukreja. R.C., Weaver, A.B. and Hess, M.L (1989) Biochim. Biophys. Aeta 990,198-205. 32 Gailis, L., Nayler, W.G. and Harding, S.E. (1990) Can. J. Physiol. Pharmacol. 68.1170-1175. 33 Krause, S.M. and Hess, M.L. (1985) J. Mol. Cell. Cardiol. 17. 523-526. 34 Kramer, J.H., Mak, i,T. and Weglicki, W.B. (1984) Circ. IRes. 55. 120-124. 35 Eley, D.W., Koreeky, B. and Hiss, H. (1989) Am. J. Physiol. 257, HI321-HI325.
Alterations in cardiac contractile proteins due to oxygen free radicals S h i n g o S u z u k i , M a s a n o r i K a n e k o , D o n a l d C. C h a p m a n
a n d N a r a n j a n S. D h a l l a
Division of Cardiot'ascular Sciences, St. Boniface General Hospital Research Centre a,,d ~'partment of Pl~'csivh gv. Faculty of Medicine, Unit'ersity of Mmlitoba. Wimupeg {Canada)
(Received 29 October 1990) Key words: ATPase.Mg2+-:ATPase,Ca2 +-stimulated:Sulfhydrylgroup: Superoxideanion; Hydrogenperoxide: Hypochlorousacid; (Rat heart myofibril) In view of the potential role of free radicals in the genesis of cardiac abnormalities under different pathophysiological conditions and the importance of contractile proteins in determining heart function, this study was undertaken to examine the effects of oxygen free radicals on the rat heart myofibrils. Xanthine plus xanthine oxidase (X + XO) which is known to generate superoxide anions ( O z ) and hydrogen peroxide (H20~), an activated species of oxygen, was found to decrease CaZ+-stimulated ATPase activity, increase MgZ+-ATPase activity and reduce sulfhydryl (SH) group contents in myofihrils; these effects were completely prevented by superoxide dismutase (SOD) plus catalase (CAT). Both HzO 2 and hypochlorous acid (HOCi), an oxidant, produced actions on cardiac myofibrils similar to those observed by X + XO. The effects of H 2 0 z and HOCI were prevented by CAT and L-methionine, respectively. N-ethylmaleimide (NEM) and 5,5'-dithiobis(2-nitrobenzoie acid) (DTNB), inhibitors of S H groups, also produced effects similar to those seen with X + XO. Dithlothreitul (DTr), a well known sulfhydryl-redueing agent, prevented the actions of X + XO, H2Oz, HOCI, NEM and DTNB. These results suggest that marked changes in myofibrillar ATPase activities by different species of oxygen free radicals may he mediated by the oxidation of S H groups.
Introduction
Oxygen free radicals have been suggested to participate in the pathophysiology of contractile failure and cellular damage due to ischemia-reperfusion injury in the heart [1-3]. Furthermore, different oxygen free radical generating systems have been shown to induce contractile failure, muscle contracture and cell damage in the myocardium [4-6]. Since the function of the heart is primarily determined by the interaction of Ca 2 + with contractile proteins as well as by the regulatory activities of sareolemma and sarcoplasmic reticulum [7,8], it is likely that adverse effects of oxygen free radicals in the heart are due to their actions on the functions of one or more of these organelles. Earlier studies in this
Abbreviations: X, xanthine; XO, xanthine oxidase; SH, sulfhydryl group; SOD, superoxidedismutase; CAT, catalase: NEM, N-ethylmaleimide: DTNB. 5,5'-dithiobis(2-nitrobenzoicacid). Correspondence: N.S. Dhalla, Division of Cardiovascular Sciences, St. BonifaceGeneral Hospital Research Centre, 351 Tache Avenue, Winnipeg,Manitoba. Canada R2H 2A6.
regard have revealed that Ca2+-transport activities of both cardiac sarcolemma [9-12] and sarcoplasmic reticulum [13,14] are depressed upon treating these membranes with oxygen radical generating systems. However, virtually nothing is known at present concerning the effects of oxygen free radicals on myofibrils, the basic contractile machinery of the myocardial cell. This study was therefore undertaken to investigate the actions of oxygen free radical generating systems on myofibrillar ATPase activities. Since sulfhydryl groups are required for the myofibrillar ATPase activities [1517] and oxygen radicals are known to oxidize sulfhydryl groups in proteins [10,18-20], it is possible that the free radical-induced changes in myofibrillar ATPase activities are associated with alterations in myofibriUar sulfhydryl group contents. Accordingly, sulfhydryl group contents were measured upon treatment of myofibrils with oxygen free radical generating systems in order to provide some information regarding the mechanism of changes observed in this study. The effects of different free radical scavengers on alterations in myofibrillar ATPase and sulfhydryl groups were also examined to verify if the observed changes are mediated through free radicals.
Materials and Methods
Materials" Xanthine. superoxide dismutase (3600 units/mg), catalase (14100 units/mg), L-methionine, DTT, ATP, DTNB, and NEM were purchased from Sigma. Xanthine oxidase (12.45 units/ml) was the product of Calbiochem. Sodium hypochlorite (NaOCI), hydrogen peroxide, and sodium dodecyl sulfate (SDS) were from Mallinckrodt, Fisher Scientific. and Bio-Rad, respectively. All chemicals used were of analytical grade.
Methods Preparation of myofibrils. Male Sprague-Dawley rats weighing approx. 200 g were used. Animals were killed by decapitation, hearts were removed, atria and any large vessels were carefully trimmed, and the ventricular tissue was processed for the isolation of myofibrils by the method of Solaro et al. [21]. The myofibrillar fraction was suspended in a final solution containing 100 mM KCI, 20 mM Tris-HCl (pH 7.0). The protein concentration was determined by the method of Lowry et al. [22] using the bovine serum albumin as a standard. Measurement of Mg2 +-A TPase and Ca2 +-stimulated A TPase activities" of myofibrils. The experimental conditions were the same as reported elsewhere [23]. Briefly, for the estimation of Mg~+-ATPase activity, myofibrils (0.5 mg protein/ml) were preincubated at 30 o C for 5 min in 1.0 ml of medium containing: 25 m M imidazole (pH 7,0), 2 mM MgCI z, 5 mM NAN3,1 mM EGTA and 50 mM KCI. Appropriate blank tubes contained all of the above with the exception of myofibrillar proteins. The reaction was started by the addition of 2 m M ATP and terminated 5 min after by the addition of 1 ml of 127o trichloroacetic acid. These samples were centrifuged and phosphate in the protein-free supernatant was determined by the method of Taussky and Shorr [24]. Total (Ca 2÷ plus Mg 2+) ATPase activity was determined in the medium containing: 25 m M im. idazole (pH 7.0), 2 mM MgC!z, 5 m M NAN3, 50 m M KCI and 0.01 m M free Ca 2÷. The concentration of free Ca 2÷ in the medium was adjusted by using EGTA as previously described [23]. Ca2+-stimulated ATPase activity was taken as the difference between the values obtained for total and Mg2+-ATPase activities. Determination of sulfhydryl content in myofibrils. Total sulfhydryl groups in myofibrils were determined with DTNB [25]. Myofibrils (0.2 mg protein/ml) were incubated in 60 mM imidazole (pH 7.4) containing 30 mM EDTA and 0.8% SDS in a final volume of 2.58 ml. The reaction was started by adding DTNB at a final concentration of 2 mM. Absorbance was measured at 412 nm 5 min after but this value did not change within 30 min of incubation. The reference cuvette contained solvents and DTNB concentrations identical to the samples, but myofibrillar proteins were omitted. After re-
cording the final absorbance, 5 mg NEM was then added to the sample and reference cuvettes; the value obtained 5 min later was subtracted from the value in the presence of DTNB. The concentration of sulfhydryl groups was calculated by using an E molar value of 13 600 [25]. Free radical-generating systems and oxidants. Superoxide anion radicals were generated by xanthine oxidase (0.03 U / m l ) reaction by using xanthine (2 mM) as a substrate. In this experiment xanthlne oxidase was dialyzed overnight against 0.01 M Tris-HCI buffer (pH 7.4) at 4 ° C in order to eliminate the contamination by (NH4)2SO4 and EDTA which are included in the commercial preparation. Subsequently, it was pretreated with 0.4 m M phenylmethylsulfonyl fluoride (PMSF) to inhibit the associated trypsin-like activity. Hydrogen peroxide was used at the concentration of 1 raM. HOCi, a well known oxidant, was prepared by vacuum distillation of NaOCI after adjusting the pH to 6.2 with dilute sulfuric acid. The concentration of HOCI was determined using a molar absorptivity coefficient of 100 at 235 n m [26,27] and was used at the concentration of 0.4 m M in this experiment. SOD (80 # g / m l ) , CAT (10 /tg/mi), and L-methionine (1 mM) were used as scavengers for 02-, H 2 0 ~, and HOCI, respectively [9,28,29]. D T T (1 mM) was also used t, obtain a protection from the effects of these activated oxygen species [10]. The combination of H202 (0.1 mM) and Fe z+ (0.1 raM) was used to generate hydroxyl radicals [9,30]. It should be pointed out that different species of radicals are formed by the oxygen radical generating systems employed in this study and different scavengers are also non specific in nature. It is therefore not our intention to emphasize the specificity of different radical generating systems or the scavengers used here. Statistical analysis. For the determination of ATPase activity and sulfhydryl group content, all assays were carried out in duplicate by using different preparations. Results were expressed as means + S.E. For statistical evaluation, Student's t test was used. P level < 0.05 was taken to reflect a significant difference between control and treated preparations. Results
Effects of xanthine plus xanthine oxidase on heart myofibrillar A TPase activities and SH group contents The effects of 2 m M X plus 0.03 U / m l XO, a predominantly superoxide anion generating system in which some H202 is also produced, on heart myofibrillar ATPase activities were examined. Fig. 1 shows the time-dependent effects on Ca 2 +-stimulated ATPase and MgZ+-ATPase activities. After 10 min incubation of mvofibrils with X plus XO, a significant difference was apparent in Mg2+-ATPase activity but Ca2+-stimulated ATPase activity was not affected. While MgZ+-ATPase
97
-~'12 ~
0-0
Co2+
"~
0-0
Mg 2 +
TABLE I
- 6
Effects of X + X O on myofibrillar ATPase activities and S H group contents
,
~10 ~--& , ~
T , 0-_____._.__.__6
•
:~ -~
~
38
.E_ +1 •
,
,
10 20 30 60 Incubotion Time (min) Fig, I. Incubation time-dependent effects of xanthine plus xanthine oxidase on heart myofibrillar ATPase activities. Myofibrils (5 mg protein/ml) were preincubated at 30°C with xanthine (2 mM) plus xanthine oxidase (0.03 U/ml) in a medium containing 50 mM KCI, l0 mM Tfis-HCI (pH %0) in a total volume I ml, and then ATPase activities were assessed. Each value is a mean±g.E, of six preparations. • P < 0.05 vs. control.
Each value is a mean+S.E, of six to nine different preparations. Myofibrils (5 mg protein/ml) were preincubated for 20 rain at 30°C in a medium containing 50 mM KCI, 10 mM Tris-HCI (pH 7.0) plus the addition, shown in a total volume I ml. Aliquots of 0.l ml samples were assayed for ATPase activities and SH group contents. Final concentrations of xanthine (XL xanthine oxidase (XOL superoxide dismutase (SOD), and catalase (CAT) were 2 mM, 0.03 U/ml, 80 tLg/ml, and tO ~g/ml, respectively. * P < 0.05 vs, control and * * P < 0.05 vs. X + XO+ SOD.
X+XO X+XO+SOD X+XO+SOD +CAT X XO SOD CAT
(~mol/mg protein/h) ~ stimulated
contents (nmol/mg protein)
4.97 + 0.16 * 5.13+0.27"
6.48:i: 0.18 * 7.60-+@29*
54.2+1.0" 59,1 ± l.l*
10.30+0.30"* 10.34+0.31 9.89 + 0.28 10.40 + 0.25 10.34_+0.41
69.5-+0.8 "* 67.2_+1.8 68.5 + 2.2 65.5 + 1.9 66.6+2.9
2.60+0.17"* 2.e0+0.23 2.88 =i=0.23 2.67 + 0.08 2.92_+0.17
0
activity d i d n o t increase further, C a 2 + - s t i m u l a t e d A T P a s e activity w a s progressively i n h i b i t e d d u r i n g a 20 to 60 rain i n c u b a t i o n period. M y o f i b r i l s i n c u b a t e d w i t h o u t X o r X O for the s a m e p e r i o d s d i d n o t s h o w a n y c h a n g e in M g 2 + - A T P a s e o r C a 2 + - s t i m u l a t e d A T P a s e activities ( d a t a n o t shown). T a b l e 1 s h o w s t h a t i n c u b a t i o n o f m y o f i b r i l s with either 2 m M X, 0.03 U / m l X O , 80 ~ t g / m i S O D , o r 10 p , g / m l C A T for 20 m i n at 3 0 ° C d i d not a f f e c t the A T P a s e activities. A c o m b i n a t i o n o f X plus X O d e p r e s s e d C a 2 + - s t i m u l a t e d A T P a s e activity a n d increased M g 2 + - A T P a s e activity b y 37 a n d 96% o f the c o n t r o l values, respectively. S O D s h o w e d a p r o t e c tive effect o n the d e p r e s s i o n in C a 2 + - s t i m u l a t e d A T P a s e activity b u t there w a s n o effect o n the increase in M g 2 + - A T P a s e activity. T h e c o m b i n a t i o n o f C A T w i t h S O D w a s m o r e effective t h a n S O D a l o n e in p r e v e n t i n g c h a n g e s d u e to x a n t h i n e plus x a n t h i n e oxidase. It m a y b e n o t e d t h a t c o n t r o l values for m y o f i b r i l l a r M g 2+A T P a s e a n d C a = + - s t i m u l a t e d A T P a s e activities a r e similar to those r e p o r t e d elsewhere [17,23]. T h e d a t a in T a b l e I also s h o w s t h a t S H g r o u p c o n tents were d e c r e a s e d b y X plus X O ; S O D s h o w e d s o m e p r o t e c t i v e effect o n S H g r o u p c o n t e n t s . T h e c o m b i n a -
tion o f c a t a l a s e w i t h S O D i n d i c a t e d c o m p l e t e p r o t e c tion f r o m the d e p r e s s i o n o f S H g r o u p c o n t e n t s inc u b a t e d with X p l u s X O . T h e i n c u b a t i o n of m y o f i b r i l s w i t h e i t h e r 2 m M X, 0.03 U / m l X O , 8 0 / ~ g / m i S O D , or 10 p , g / m i C A T for 20 rain at 3 0 ° C d i d n o t affect the S H g r o u p c o n t e n t s . In o r d e r to test the role o f c h a n g e s in S H g r o u p c o n t e n t s in X p l u s X O i n d u c e d a l t e r a t i o n s in m y o f i b r i l l a r A T P a s e activities, the effects o f different c o n c e n t r a t i o n s o f D T T w e r e e x a m i n e d . T h e results in Fig. 2 i n d i c a t e t h a t a significant p r o t e c t i v e a c t i o n of D T T o n the d e p r e s s a n t effect o f X p l u s X O for C a -'+-
r c2
4~
!li!ii!!H
0 on rol
100 1000 0 Concentrationof DTT iuM) Fig. 2. Dose-dependent effects of dithiothreitol (DTT) on myofibrillar ATPase activities in the absence (control) or presence of xanthine (X) plus xanthin¢ oxidase (XO). Myofibrils (5 mg protein/nil) were preincubated for 20 rain at 30* C with various concentrations of DTT with or without 2 mM X plus 0.03 U/ml XO in a medium containing 50 mM KCI and 10 mM Tris-HCI (pH 7.0). Each value is a mean :t: S.E. of four different preparations. * P < 0.05 vs. 0 iX plus XO without DTr).
s t i m u l a t e d A T P a s e activity w a s evident at 10 # M c o n c e n t r a t i o n ; m a x i m a l a c t i o n w a s seen at 50 p M c o n c e n t r a t i o n o f D T T . O n the o t h e r h a n d , X + X O i n d u c e d increase in M g 2 + - A T P a s e w a s significar~tly p r o t e c t e d at 50 ttM c o n c e n t r a t i o n ; c o m p l e t e p r o t e c t i o n w a s seen a t 100 # M D T T .
Effects of hydrogen peroxide and hypochlorous acid on A TPase actioities and S H group contents T h e results in T a b l e II i n d i c a t e t h a t m y o f i b r i l l a r C a 2 + - s t i m u l a t e d A T P a s e activity a n d S H g r o u p c o n tents were decreased w h e r e a s M g 2 + - A T P a s e activity w a s increased b y 1 m M H 2 0 2. T h e s e c h a n g e s in myofibrils were p r o t e c t e d b y 10 t t g / r n l C A T . T h e inhibition o f C a 2 + - s t i m u l a t e d A T P a s e activity a n d the s t i m u l a t i o n of M g 2 + - A T P a s e activity b y H 2 0 2 w e r e
~.}'~[ "e~+-~,,mo,ot,,,, 0 M ~ "+
16
Control
DTN8
il
DTNB NEM NEId DTT DTT Fig. 3. Effects of SH reagents on myofibrillar ATPase activities. Myofibrils (5 mg protein/ml) were preincubated for 20 min at 30°C with 0.1 mM DTNB or 0.05 mM NEM in the absence or presence of 1 mM DTT in a medium containing 50 mM KCI and 10 mM Tris-HCI (pH 7.0). Values are means:l:S.E, of five to eight different preparations. * P < 0.05 vs. control and *P < 0.05 vs. DTNB or NEM.
TABLE 11
Effects of H20, on myoflbrillarATPase activities and S H group contents Each value is a mean+S.E, of four to five different preparations. Myofibrils (5 mg protein/ml) were preineubated for l0 rain at 30 o C in a medium containing 50 mM KCI, l0 mM Tds-HCI (pH 7.0) in the absence or presence of different interventions indicated below. Final concentrations of H202, catalase, and DTT were 1 raM, 10 #g/ml, and 1 raM, respectively. * P < 0.05 vs. control and * * P < 0.05 vs. H 202 .
Control HzOz H202 +CAT H202 + DTT
ATPase activity (ttmol/mg protein/h) Mg2+ Ca 2+. stimulated
SH group contents (nmol/mg protein)
2.96 + 0.l 2 5.464-0.18" 2.924-0.16"* 2.684-0.13"*
66.3 :l: 2.7 47.6:1:2.6" 65.84-2.3**
10.89:1:0.45 5.92:1:0.38" 10.404-0.53"* 10.194-0.41"*
TABLE II1
Effects of HOCI on myofibrillar ATPase activities and SH group contents Each value is a mean+S.E, of five to six different preparations. Myofibrils (5 mg protein/ml) were preincubated for 10 rain at 30°C with or without various additions in a medium containing 50 mM KCI, 10 mM Tris-HCI (pH 7.0). Final concentrations of hypochlorous acid (HOCI), L-methionine (Meth), and DTT were 0.4 raM, l raM, and 1 mM, respectively. * P < 0.05 vs. control and ** P < 0.05 vs. HOCI.
Control HOCI HOCI+ Meth HOCI+ DTI" Meth DTT
ATPase activity (/~mol/mg protein/h) Mg2 + Ca2+. stimulated
SH group contents (nmol/mg protein)
2.80+0.12 9.51 +0.16 * 3.79+0.16"* 3.55+0.14"* 2.80+0.11 2.80 :l:0.13
67.6+1.1 35.7+ 2.9 * 59.84-0.9** 67.24-0.4
10.96+0.15 5.73:1:0.31* 11.644-0.12"* 11.09+0.19"* 11.20+0.16 11.034-0.19
s i g n i f i c a n t l y b l o c k e d b y 1 m M D T T . T o assess the effects o f H O C I o n A T P a s e activities a n d S H g r o u p c o n t e n t s , m y o f i b r i l s w e r e i n c u b a t e d for 10 r a i n at 30 o C with this o x i d i z i n g a g e n t ( T a b l e III). H O C 1 (0.4 m M ) d e p r e s s e d b o t h C a 2 + - s t i m u l a t e d A T P a s e activity a n d S H g r o u p c o n t e n t s b u t i n c r e a s e d the M g 2 + - A T P a s e activity. T h e s e c h a n g e s w e r e s i g n i f i c a n t l y p r e v e n t e d b y 1 m M L - m e t h i o n i n e . H O C I i n d u c e d c h a n g e s in M g 2+A T P a s e a n d C a 2 + - s t i m u l a t e d A T P a s e w e r e also p r e v e n t e d b y 1 m M D T T . It c a n b e seen f r o m T a b l e III that L-methionine alone did not show any significant effects o n A T P a s e activities o r S H g r o u p c o n t e n t s . Likewise, D T T a l o n e d i d n o t a f f e c t the m y o f i b r i l l a r A T P a s c a,;fivities. It s h o u l d also b e m e n t i o n e d t h a t e x p e r i m e n t s to test the effects o f h y d r o x y l radicals, w h i c h a r e p r o d u c e d b y F e 2+ p l u s l o w c o n c e n t r a t i o n s o f H 2 0 2 , c o u l d n o t b e t r i e d b e c a u s e 0.1 m M F e 2+ h a s a m a r k e d a c t i o n in i n c r e a s i n g a n d d e p r e s s i n g the m y o f i b r i l l a r M g 2 + - A T P a s e a n d Ca2 +-stimulated A T P a s e activities, respectively ( d a t a n o t shown).
Effects ojo~ ffhydryl reagents on M g 2 +- and C a : +-stimulated A T r , ~ e activities T o e x a m i n e the role o f S H g r o u p s in a l t e r i n g the m y o f i b r i l l a r A T P a s e activities, the effects o f D T N B a n d N E M , well k n o w n i n h i b i t o r s o f S H g r o u p s , w e r e inv e s t i g a t e d u n d e r the e x p e r i m e n t a l c o n d i t i o n s e m p l o y e d in this s t u d y (Fig. 3). C a 2 + - s t i m u l a t e d A T P a s e activity w a s depres,,~d w h e r e a s M g Z + . A T P a s e activity w a s increased b y 0.1 m M D T N B o r 0.05 m M N E M . D T T (1 m M ) s h o w e d p r o t e c t i v e effects o n the c h a n g e s in m y o f i b r i l s d u e to D T N B o r N E M . Discussion In this s t u d y w e h a v e s h o w n t h a t t r e a t m e n t o f h e a r t m y o f i b r i l s w i t h X p l u s X O a n d H2Oz d e c r e a s e d the
99 Ca2+-stimulated ATPase activity and incre:~sed the Mg2÷-ATPase activity. Since the effects of X pius XO were completely prevented by a combination of both SOD and CAT and those of H202 were completely prevented by CAT, it is evident that these actions of X plus XO were mediated by the generation of both superoxide anion radicals and H202 in the incubation medium. The observed decrease in Ca2+-stimulated ATPase activity l.~y X plus XO does not seem to be due to corresponding increase in Mg'-+-ATPase activity because (i) a significant increase in Mg 2 +-ATPase activity was not associated with a significant decrease in Ca -'+stimulated ATPase activity upon incubating myofibrils with X plus XO for a period of 10 min, (ii) a marked depression in Ca2+-stimulated ATPase was not associated with a corresponding increase in Mg2+-ATPase activity during 20 to 60 min incubation period of myofibrils with X plus XO, (iii) a significant protection of X plus XO induced decrease in Ca'-+-stimulated ATPase activity by SOD was not associated with a significant protection of X plus XO induced increase in Mg2+-ATPase activity, and (iv) a significant prevention of X plus XO induced decrease in CaZ+-sdmulated ATPase activity was seen at 10 ~tM concentration of D T T whereas 50 # M concentration of DTT was required to demonstrate a significant effect on the changes in Mg'-+-ATPase. In view of the fact that Ca "-+ sensitivity (Ca2+-stimulated ATPase) and basal activity (Mg2+-ATPase) of the myofibriUar ATPase system are manifested due to the interaction of several components such as aetin, myosin, troponin and tropomyosin [15], it is possible that the observed effects of superoxide anions and H20'- are due to their actions on one or more contractile and regulatory proteins. Extensive studies in this regard are required to establish the exact site of action of the oxy radicals on myofibrils. HOCI, which is gererated as an oxidant upon activation of neutrophils [2' ], was found to increase Mg 2+ATPase and decrease the Ca2+-stimulated ATPase activities in rat heart myofibrils. Other oxidants, such as diamide, have also been shown to exert similar effects on the guinea pig heart myofibrillar preparations [32]. The effects of HOCl on myofibrils were prevented by L-methionine, a well known scavenger of this oxidizing agent [31]. Since the actions of HOCI and diamide on myofibrillar ATPases were similar to those observed with superoxide anions and H'-O'-, it is likely that the effects of free radicals are mediated due to oxidative stress on myofibrillar proteins. Although oxygen free radicals and HOCI have also been reported to inhibit several membrane bound enzymes such as sarcoplasmic reticnlar CaZ+-stimulated ATPase as well as sarcolemmal N a + / K ÷ - A T P a s e and Ca'-÷-stimulated ATPase [9,10,12,20,31,33,34], these studies do not provide any evidence as to whether the inhibitory effects on enzyme activities are due to peroxidation of the membrane
lipids or direct oxidation of the membrane proteins. Since the myofibrillar preparation employed in our experiments is devoid of any associated lipids [21], it is reasonable to conclude that the observed effects of free radicals and HOCI are due to direct oxidation of myofibrillar proteins. The experiments reported in this study reveal that changes in myofibrillar ATPase due to superoxide anions, H202 and HOCI were associated with reduction in myofibrillar SH group contents. Furthermore, respective scavengers of these free radicals and oxidants were able to prevent these alterations in myofibrillar SH group contents. In view of a crucial role of SH groups in the expression of myofibrillar ATPase activities [1517], it is possible that the observed effects of free radicals and oxidants on myofibrillar ATPases are due to oxidation of SH groups. This view is supported by the fact that DTT, a well known sulfhydryl reducing agent, was able to prevent changes in myofibrillar ATPases due to X plus XO, H202 and HOCL Furthermore, both DTNB and NEM, well known inhibitors of SH groups [10,17], were found to increase Mg2 +-ATPase and decrease Ca2+-stimulated ATPase activities in myofibrils. It should also be noted that the actions of both DTNB and N E M like those seen with radicals and oxidants were prevented by DTT. It is therefore suggested that the observed effects of oxygen free radicals, H202 and HOCI on myofibrillar ATPase activities are mediated by the oxidation of myofibrillar SH groups, Since oxygen radical species have also been shown to affect amino acids, protein-protein erosslinking, and protein strand scission [18,19], it is not our intention to rule out the actions of free radicals on these sites. Nonetheless, the observed effects of X plus XO, H202 and HOCI on myofibrillar ATPase can be seen to explain the contractile dysfunction of myocardium by these interventions [4-6,35]. The involvement of myofibrillar defects due to the action of free radicals and oxidants in altering the contractile function may not be specific in nature because these interventions have also been reported to affect sarcolemmai and sareoplasmic reticular functions under similar experimental conditions [9-14]. Acknowledgments The work reported in this study was supported b9 a granz from the Medical Research Council of Canada. Dr. S. Suzuki was a postdoctoral fellow of the Medical Faculty at the University of Manitoba during the tenure of this study. References 1 Ambro~io.G. Pecker. L.C- Hulchins. G.M.. Welsman.H.F. and Weisfeldt. M.L. (1986) Circulation 74.1424-1433.
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