© 1996 American Heart Association, Inc.
Articles |
Creatine Kinase Is the Main Target of Reactive Oxygen Species in Cardiac Myofibrils
From Cardiologie Cellulaire et Moléculaire (H.M., V.V., P.M., R.V.-C.), U-446 INSERM, Université Paris-Sud, Faculté de Pharmacie, Châtenay-Malabry, France, and Laboratoire de Physiopathologie et Pharmacologie Cardiovasculaires Experimentales (V.M., L.R.), Université de Bourgogne, Faculté de Médecine, Dijon, France.
Correspondence to Dr R. Ventura-Clapier, Cardiologie Cellulaire et Moléculaire, U-446 INSERM, Université Paris-Sud, Faculté de Pharmacie, 5 rue J-B Clément, 92 296 Châtenay-Malabry, France.
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Abstract |
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Abstract Reactive oxygen species (ROS) have been reported to alter cardiac myofibrillar function as well as myofibrillar enzymes such as myosin ATPase and creatine kinase (CK). To understand their precise mode and site of action in myofibrils, the effects of the xanthine/xanthine oxidase (X/XO) system or of hydrogen peroxide (H2O2) have been studied in the presence and in the absence of phosphocreatine (PCr) in Triton X-100–treated cardiac fibers. We found that xanthine oxidase (XO), with or without xanthine, induced a decrease in maximal Ca2+-activated tension. We attributed this effect to the high contaminating proteolytic activity in commercial XO preparations, since it could be prevented by a protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), and it could be mimicked by trypsin. In further experiments, XO was pretreated with 1 mmol/L PMSF. Superoxide anion production by the X/XO system, characterized by electron paramagnetic resonance spin-trapping technique, was not altered by PMSF. A slight increase in maximal force was then observed either with X/XO (100 µmol/L per 30 mIU/mL) or H2O2. pMgATP–rigor tension relationships have been established in the presence and in the absence of PCr to separate the effects of ROS on myosin ATPase and myofibrillar-bound CK. In the presence of PCr, pMgATP50, the pMgATP necessary to induce half-maximal rigor tension, was reduced from 5.03±0.17 (n=21) to 4.22±0.22 (n=4) after 25 minutes of incubation in the presence of 30 mIU/mL XO and 100 µmol/L xanthine or to 4.04±0.1 (n=11) after incubation in the presence of 2.5 mmol/L H2O2. The ROS effects were partially prevented or antagonized by 1 mmol/L dithiothreitol. No effect was observed on pMgATP50 when PCr was absent. pCa-tension relationships have been evaluated to assess the effects of ROS on active tension development. Incubations with H2O2 induced an increase in Ca2+ sensitivity and resting tension when MgATP was provided through myofibrillar CK (PCr and MgADP as substrates) but not when MgATP was added directly. These results suggest that myofibrillar CK was inhibited by ROS. Active stiffness and the time constant of tension changes after quick stretches applied to the fibers were dose-dependently increased by H2O2 only in the presence of PCr. In addition, myofibrillar CK but not myosin ATPase enzymatic activity was depressed after incubation with either ROS. These results suggest that ROS mainly alters CK in myofibrils, probably by the oxidation of its essential sulfhydryl groups. Such CK inactivation results in a decrease in the intramyofibrillar ATP-to-ADP ratio. The effects of ROS on cytosolic and bound CKs may take part in the overall process of myocardial stunning after cardiac ischemia and reperfusion.
Key Words: skinned fibers • myofibrillar creatine kinase • active tension • crossbridges • rigor tension
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Introduction |
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Oxygen-derived free radicals have been implicated in various types of myocardial injury, especially in ischemia/reperfusion injury (for review, see References 1 and 21 2 ). Electron spin resonance investigations have demonstrated that a brief episode of ischemia and the subsequent reperfusion are associated with free radical generation.3 4 5 In reperfused hearts, it has been shown that both the decrement of oxygen-derived free radicals after the initial burst and the recovery of the stunned myocardium are accelerated by agents that either scavenge oxygen radicals, such as SOD and CAT,6 7 or prevent their generation, such as allopurinol and desferrioxamine.8 Alterations in the recovery of PCr, following the peak of oxygen radical production, were shown to be largely prevented by the administration of SOD.9
Different ROS have been shown to induce negative inotropic effects, contracture, cell damage, an increase in lipid peroxidation, and a decrease in high-energy phosphate contents in the myocardium10 11 12 (for review, see Reference 11 ). Since the contractile apparatus plays the central role in cardiac function, deleterious effects of ROS on myofibrils would be of critical importance. Thin and thick filaments contain SH groups that could be modified by oxidizing agents. The main myofibrillar enzymatic systems, myosin ATPase and CK, have been shown to be susceptible to different ROS. The marked changes in myofibrillar ATPase and CK induced by X/XO and hydrogen peroxide (H2O2) may be mediated by the oxidation of SH groups in the enzyme.13 14 15 Incubation of cardiac myofibrils with diamide (an oxidizing agent), H2O2, X/XO, or hypochlorous ions (HOCl) increases basal ATPase activity and abolishes its Ca2+ dependence.13 16
Skinned fibers have been used to assess the consequences of myofibril exposure to different ROS on the intrinsic mechanical properties of contractile proteins. ROS have been shown to induce quite specific alterations, the mechanisms of which have not been identified. A sharp and irreversible decline in maximal force, without changes in Ca2+ sensitivity, has been described with the X/XO system.17 It was shown that HOCl mainly increased rigor tension and the Ca2+ sensitivity of active tension, whereas H2O2 only marginally affected mechanical properties.18
The aim of the present study was to characterize the functional consequences of the exposition of myofibrillar proteins to ROS. Triton X-100–treated cardiac fiber bundles were used to assess intrinsic mechanical properties of myofibrils in controlled ionic and substrate environment. By studying the action of ROS in the presence or in the absence of PCr, we sought to compare the susceptibility of myofibrillar CK and myosin ATPase. MgATP was provided either directly in the solution or through endogenous ATP production catalyzed by myofibrillar-bound CK in the presence of PCr and MgADP. The results show that the main target of ROS in myofibrils seems to be the essential SH groups of CK. Oxidation of these groups would induce an inhibition of the enzyme and an alteration of the contractile properties as a consequence of a decreased ATP-to-ADP ratio in the myofibrillar compartment.
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Materials and Methods |
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Mechanical Experiments
Muscle Preparation
Rats were anesthetized with an intraperitoneal injection of urethane (2 g/kg) according to the recommendations of the institutional Animal Care Committee (INSERM, Paris, France). Muscle fiber bundles (
200 µm in
diameter) were dissected from papillary muscles of the left ventricle
of rats in a zero-Ca2+ Krebs' solution, pH 7.4. Fibers
were incubated for 1 hour in a relaxing solution (solution A; see
solutions below) containing 1% Triton X-100 to solubilize the
membranes and were then transferred to the relaxing solution without
detergent. After the skinning procedure, the fiber was mounted in the
experimental apparatus. It was adjusted to slack length,
stretched by 20%, and subjected to an activation/relaxation cycle.
Sarcomere length was adjusted to 2.2 µm using laser diffraction (10
mW He-Ne laser, Spectra Physics Inc) after the first activation cycle.
The length and diameter of the muscles were measured using a graticule
in the dissecting microscope. Muscles were immersed in 2.5-mL chambers
arranged around a disk in a temperature-controlled bath positioned
on a magnetic stirrer. Each solution was well stirred at high speed
(>1000 rpm). All experiments were performed at 22°C.
Experimental Apparatus
One fiber was snared at both ends with hair emerging from
stainless steel tubes, between a transducer (model AE 801, SensoNor
Microelectroniks) and a vibrator.19 The bandwidth of the
transducer and tube was 2 kHz. The permanent magnet and coil came from
a standard loudspeaker (Pioneer TS-130A, Pioneer Electric and Research
Corp). The coil was glued to a glass tube axis (2 mm in diameter)
driven in an axial ball bearing (total moving mass, <1.5 g). A flag
with a narrow window was glued on the glass axis between a lamp and a
position detector (type S1543, Hamamatsu), allowing measurement of the
displacement length. A feedback with the length signal combined with a
power amplifier allowed control of muscle length. The system had a rise
time of 1 millisecond without overshoot. Length and tension changes
were driven by a pulse generator (GP1-GE 219, CEA-DAM France) and
monitored on an oscilloscope (model OS4020, Gould, Inc). Tension and
length tracings were digitized at 20 kHz (12-bit
analog-to-digital converter), analyzed on-line
using a computer (Deskpro 486, COMPAQ Corp), and stored on a
videotape.
Solutions
Solutions were calculated using the computer program of
Fabiato20 and are listed in Table 1
. Rigor
solutions were obtained by mixing solutions A4 and E' with
PCr or solutions C and G without PCr. Activating solutions were
obtained by mixing two solutions of pCa 9 and 4.5 (solutions A and B,
respectively). EGTA, PMSF, xanthine,
H2O2, CAT, SOD, DMPO, and
desferrioxamine were purchased from Sigma Chemical Co. XO (X4376) was
obtained from Sigma as lyophilized compound. PCr (Neoton, Schiapparelli
Farmaceutica) was a kind gift of Prof E. Strumia. All solutions for ROS
generation were prepared immediately before use. Experiments were
performed at pH 7.1 and 22°C.
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Experimental Protocol
pMgATP–Rigor Tension Relationships
Before and after incubating the fiber in solution A containing
either the X/XO system or H2O2 for 25 minutes,
pMgATP–rigor tension relationships were established by a stepwise
decrease in ATP concentration until maximal rigor tension was obtained.
Data were fit using the Hill equation:
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pCa-Tension Relationships
Resting tension was the tension in relaxing solution A at a
sarcomere length of 2.2 µm. Tmax of each fiber was obtained at pCa
4.5. The pCa-tension relationships were established under isometric
conditions by stepwise increasing [Ca2+] until Tmax was
reached. Data were fit using the Hill equation:
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Stiffness and Kinetic Measurements
Fiber stiffness was estimated by the quick length change
technique as previously described.19 A series of seven
stretches and five releases of 0.3% to 3% initial muscle length were
applied in relaxing or activating solutions. Only responses to stretch
were used for calculations. The spike of tension in phase with the
length change characterizes the elastic phase.21 Stiffness
was the extreme tension reached during stretch (in millinewtons per
square millimeter) divided by the length change (in
micrometers). Each value for each fiber is the mean of five
to seven determinations after stretches of varying amplitudes in a
given experimental condition. A first series of length changes was
imposed in the relaxing solution to assess the passive properties of
the fiber. Resting stiffness was calculated by linear regression
analysis on the responses to stretches. Then a second series of
length changes was initiated in the control activating solution. The
tension level before the first stretch was taken as the Tmax and was
used for normalization. Active stiffness was calculated as the
difference between total stiffness and resting stiffness.
The spike of tension occurring after a quick stretch is followed by a rapid drop in tension to a minimum value. The time constant (in milliseconds) of this tension drop was calculated by a least-squares regression analysis.
Enzyme Activity Measurements
CK and myosin ATPase activities were measured according to
Hoerter et al22 on skinned isolated cells in conditions
similar to those for the mechanical experiments.
Adult rat cardiac myocytes were isolated by collagenase
perfusion according to Pucéat et al.23 After
isolation, the cardiomyocytes were skinned for 5 minutes in
solution A containing 0.5% Triton X-100 and washed twice. Freshly
isolated skinned cells were incubated for 25 minutes at 22°C in
solution A (3 mg protein per milliliter) in the presence or in the
absence of either H2O2 or the X/XO system.
After incubation, aliquots were used for the assays.
CK activity was assayed using the coupled-enzyme system of G-6-PDH
and hexokinase. CK activity was determined by measuring NADPH
production at 340 nm (Gilford Spectrophotometer) in solution E'
(Table 1
) also containing (mmol/L) ADP 1, glucose 20, and NADP 0.6,
along with 2 IU/mL of both hexokinase and G-6-PDH at a pH 7.1 and
22°C. Reaction was initiated by PCr addition.
Myosin ATPase activity was assayed using the coupled-enzyme system
of pyruvate kinase and lactate dehydrogenase. ATPase activity was
determined by measuring NADH consumption at 340 nm in solution C (Table 1) containing, in addition, 0.5 mmol/L phosphoenolpyruvate, 0.15 mmol/L
NADH, and 2 IU/mL of both pyruvate kinase and lactate dehydrogenase at
pH 7.1 and 22°C. Sodium azide (5 mmol/L) was added to inhibit
possible residual mitochondrial ATPase activity. The Mg-ATPase reaction
was started by addition of ATP. Ca,Mg-ATPase activity was measured
after the addition of CaCl2 to obtain pCa 4.5. Ca-ATPase
activity was the difference between Mg-ATPase and Ca,Mg-ATPase
activities. The protein content of skinned isolated cells was
determined by the Lowry assay.24
Free Radical Generation
Free radical species generated by the different systems used
were characterized by EPR spin-trapping techniques using the spin trap
DMPO at a final concentration of 50 mmol/L. DMPO was further purified
by double distillation. Care was taken to keep the DMPO solution
protected from light-induced degradation.
EPR spectra were recorded in a flat-type quartz cell at room temperature with an X-band Bruker ESP 300E spectrometer with a TM110 cavity using a modulation frequency of 100 kHz, modulation amplitude of 0.5 G, microwave power of 20 mW, time constant of 0.16 second, and a scan rate of 100 G/42 s. Results are expressed in arbitrary units.
Statistical Analysis
Values are expressed as mean±SEM. Student's t test
was used to compare the means. Statistical significance was reached at
P
.05. Linear regression analysis was carried out
using the least-squares method.
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Results |
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Effects of H2O2 and X/XO on Maximal Ca2+-Activated Tension
The effects of H2O2 on Tmax were studied after activating the fiber at pCa 4.5. As shown in Fig 1
, increasing the concentration of
H2O2 from 0.5 to 5 mmol/L induced a slight and
irreversible rise in Tmax. After relaxing the preparation in the
absence of H2O2, the resting tension
failed to return to the control level, so that active tension per se
diminished.
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The X/XO system is widely used to produce specific ROS, mainly
superoxide anion (O2-
) and
H2O2. We found that X/XO, in contrast to
H2O2, induced an irreversible slow
decline in Tmax, followed by a complete recovery of resting tension as
previously described.17 This decline was accelerated by
increasing activities of XO with a threshold for 2 to 3 mIU/mL. When XO
was applied without xanthine, Tmax nevertheless decreased. DTT (1 or 2
mmol/L), a reducing agent, did not prevent or block the decrease. When
the fiber was incubated in the presence of 400 IU/mL SOD, which
catalyzes the dismutation of superoxide anions into
H2O2, together with 100 IU/mL CAT, which
reduces H2O2 into H2O, further
application of 50 mIU/mL XO alone still induced the same decrease in
maximal force, whereas further addition of the substrate (xanthine, 50
µmol/L) did not change the slope of the tension fall until complete
disappearance of force (Fig 2A).
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Mammalian xanthine dehydrogenase/XO is an interconvertible enzyme, and
the conversion from xanthine dehydrogenase to XO occurs either
reversibly by oxidation of the SH residues or irreversibly by
proteolysis.25 Commercial preparations of XO thus contain
a contaminating proteolytic activity.13 In order to see
whether a proteolytic activity can mimic the XO effects on Tmax, we
applied 10 mIU/mL trypsin to a maximally
Ca2+-activated fiber. Trypsin induced a fall in
Tmax similar to that induced by XO, which could be inhibited by a
specific inhibitor of trypsin-like proteases, leupeptin
(10 and then 20 µmol/L, Fig 2B). Leupeptin did not completely inhibit
the decrease in Tmax induced by XO. Yet, another protease
inhibitor, PMSF, could prevent the decrease. When 1 mmol/L
PMSF was applied, tension first abruptly decreased and then stabilized,
but further addition of XO (50 mIU/mL) with or without xanthine (50
µmol/L) did not induce further decline; moreover, PMSF could stop the
decline induced by XO (50 mIU/mL) alone (Fig 2C). Finally, since PMSF
alone induced a sharp and small decrease in Tmax, the stock solution of
XO was pretreated with 1 mmol/L PMSF to completely block the protease
activity, so that the final concentration of PMSF applied to the fiber
never exceeded 10 µmol/L. Application of PMSF-pretreated XO (30
mIU/mL) alone was without effect on Tmax (Fig 2D). Utilization of
pretreated XO in the presence of xanthine did not induce any decrease
in Tmax, but a small increase was generally observed for high X/XO
activity (100 µmol/L per 30 mIU/mL). In the following experiments,
pretreated XO was always used.
Free Radical Generation
Since many substances have been described to exert free
radical–scavenging effects, we analyzed the
production of free radicals by the X/XO system and
H2O2 in the solutions used for skinned fiber
studies and in the presence or absence of PMSF to ensure that this
molecule did not act as a free radical scavenger.
As shown in Fig 3a, the EPR spectrum obtained with the
X/XO system in solution A consisted of a prominent 12-line signal
arising from the DMPO-superoxide adduct, DMPO-OOH, and a smaller
quartet signal originating from the hydroxyl adduct, DMPO-OH. The
DMPO-OH signal could be due to the decomposition of DMPO-OOH or to
direct trapping of · OH radicals. The addition of ethanol (10%
[vol/vol]), which could potentially react with ·OH radicals and
give ·CH(OH)CH3 radicals, was not associated with the
appearance of the DMPO-CH(OH)CH3 adduct, suggesting that
under our experimental conditions, DMPO-OH generation occurred mostly
from the breakdown of DMPO-OOH rather than from direct ·OH trapping.
PMSF at a final concentration of 10 µmol/L, used in the mechanical
studies, did not modify the EPR spectrum and therefore did not show any
superoxide-scavenging property (Fig 3b). However, SOD (10 IU/mL)
scavenged the superoxide radicals, preventing the formation of DMPO
adducts (Fig 3c). The superoxide generation continued for 10 minutes
and was 8.4 arbitrary units per milliliter per minute (10 nmol
per milliliter per minute) in the presence of 30 mIU/mL XO and 100
µmol/L xanthine. With the lower xanthine concentration (30 µmol/L),
the superoxide generation intensity was reduced to 1.8 arbitrary
units per milliliter per minute but was prolonged until 25 minutes (Fig 3d). In solution A, H2O2 used at a
concentration of 2.5 mmol/L in the mechanical studies did not induce
the formation of a DMPO adduct, even when the DMPO concentration was
increased to 100 mmol/L, suggesting the absence of or very low free
radical generation.
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Effects of H2O2 or X/XO on Rigor
Tension Development
In order to elucidate the mechanisms by which free radicals alter
the myofibrillar function, we first studied the effects of ROS on rigor
tension development. Rigor tension develops in the absence of
Ca2+, when MgATP is depleted close to the myosin ATPase. We
have shown previously that when PCr is present,
myofibrillar-bound CK can increase the local ATP-to-ADP ratio, and
the pMgATP–rigor tension curve is shifted toward a lower MgATP; the
amplitude of the shift is thus an estimate of the myofibrillar CK
efficacy (for review, see Reference 2626 ). Since the ROS are potentially
able to inhibit both myosin ATPase and myofibrillar CK, the effects of
H2O2 and of the X/XO system on rigor tension
development were assessed in the presence and absence of PCr to
dissociate their influence on either myosin ATPase or CK function in
myofibrils.
In the following experiments, fibers were incubated with either ROS for 25 minutes in the relaxing solution to compare their long-lasting effects on rigor tension. pMgATP–rigor tension relationships were obtained by bathing the fibers in a set of solutions of decreasing MgATP before and after exposure to the ROS.
Effects of 2.5 mmol/L H2O2 on typical
fibers are shown in Fig 4. In the absence of PCr, the
threshold for rigor tension development was in the millimolar range,
and the MgATP concentration for half-maximal rigor tension was
400 µmol/L (Fig 4A). Incubation of the fiber with
H2O2 was without effect on rigor tension
development.
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indicates
control curve; 
, after incubation with
H2O2. Resting tension was 2.7
mN/mm2; active tension was 27.7 mN/mm2. B, In
the presence of 12 mmol/L PCr, the relationship was shifted toward
lower MgATP concentrations (control curve [
]). The fiber was
then successively submitted to different incubations. After a 25-minute
incubation with 2.5 mmol/L H2O2 and 100 IU/mL
CAT (Cat) to degrade H2O2 (
), no effect
was observed. The fiber was then incubated for 25 minutes with 1 mmol/L
DTT together with 2.5 mmol/L H2O2 (x). After a
25-minute incubation with H2O2 alone (
) partially reversed the effects of
H2O2. Resting tension was 2.6
mN/mm2; active tension was 53.2 mN/mm2.
In the presence of PCr, under control conditions, rigor tension
developed at much lower MgATP concentrations, with an MgATP
concentration for half-maximal tension of 10 µmol/L, as
previously described.26 27 28 Fig 4B shows the behavior of a
representative fiber. After the control pMgATP–rigor
tension relationship in the presence of PCr had been obtained, the
fiber was first incubated for 25 minutes in the presence of 2.5 mmol/L
H2O2 and CAT (100 IU/mL). No shift in the
pMgATP–rigor tension relationship was observed, showing that CAT
completely prevented the effects of H2O2. When
the incubation was performed without CAT but in the presence of 2.5
mmol/L H2O2 together with 1 mmol/L DTT (a
concentration used to prevent myosin ATPase and CK inhibition by XO,
H2O2, or HOCl13 14 15 ), the
pMgATP–rigor tension curve was shifted slightly to the right. However,
when this fiber was then incubated in the presence of
H2O2 alone, the shift was much more pronounced,
reaching a value close to control conditions in the absence of PCr (see
Table 2 for mean values). Finally, in the presence of 1
mmol/L DTT, the curve returned to the level attained after incubation
in the presence of both H2O2 and DTT. These
results suggest that not all the effects of
H2O2 could be attributable to DTT-sensitive SH
group oxidation and that this long protocol did not significantly alter
the fiber response. Effects of H2O2 on rigor
tension development in the presence of PCr were not prevented by the
addition of 100 µmol/L desferrioxamine, a concentration sufficient to
inhibit iron-mediated CK inactivation29 (data not
shown), suggesting that hydroxyl radicals were not involved. Exposure
of the fibers to lower H2O2 concentration (100
µmol/L) for up to 25 minutes had no effect on resting tension or
rigor tension with or without PCr (data not shown).
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The same kinds of experiments were performed with the X/XO system (Fig 5 and Table 2). Results similar to the
H2O2 effects were obtained. X/XO incubation did
not alter rigor tension development in the absence of PCr (Fig 5A). In
the presence of PCr (Fig 5B), incubation with X/XO in the presence of
400 IU/mL SOD and 100 IU/mL CAT was without effects. X/XO alone induced
a rightward shift of the pMgATP–rigor tension relationship, which
could be partially reversed by 1 mmol/L DTT. Increasing DTT
concentration to 2 mmol/L did not further modify rigor tension (data
not shown). When the fibers were incubated with 3 mIU/mL pretreated XO
and 30 µmol/L xanthine, no effects on rigor tension were
observed.
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). After a 25-minute incubation
with XO/X alone (Since the ROS were effective only when PCr was present, the results suggest that the alterations in rigor tension development are mainly the consequence of an inhibition of myofibrillar CK.
Effects of H2O2 on Active Tension
Development
ROS have been shown to increase Ca2+ sensitivity of
tension development.18 Ca2+ sensitivity is a
complex parameter that depends on many factors, such as
regulatory proteins on the thin filament, protein interactions within
the thin filament, and the kinetics of crossbridges. In order to
identify the possible target and to dissociate between the direct
effects of ROS on the thin-filament regulatory proteins and the
indirect effects via myosin ATPase or CK alterations, Ca2+
sensitivity of skinned fibers was assessed by stepwise increasing the
Ca2+ concentration in the presence of different substrates.
One set of solutions (solutions C and D in Table 1) contained MgATP
(3.16 mmol/L) as a substrate, whereas in the other set (solutions E and
F in Table 1), MgATP was provided exclusively through the
rephosphorylation of MgADP by the
endogenous CK in the presence of PCr. When MgATP was the
only substrate, the pCa-tension relationships obtained before and
after incubation of a representative fiber with 2.5
mmol/L H2O2 for 25 minutes were similar (Fig 6A). The averaged pCa50 and Hill coefficient
were, respectively, 5.98±0.13 and 1.44±0.08 (n=3) before and
5.96±0.13 and 1.27±0.06 (n=3) after incubation. As already
described,28 when MgATP was provided by the CK reaction,
the pCa-tension relationship was shifted toward higher
Ca2+ concentrations, and the slope became more steep
(pCa50, 5.53±0.04 [n=3, P<.05];
nH, 2.63±0.25 [n=3]; P<.05 compared
with MgATP alone). After incubation with
H2O2, a high increase in resting tension
was observed together with an increase in Ca2+ sensitivity
(Fig 6B). After normalization, the relationship appeared shifted toward
lower Ca2+ concentrations (pCa50,
6.07±0.19 [n=3]; P<.01), and Hill coefficient decreased
to 1.03±0.21 (n=3, P<.01). Since changes in
Ca2+ sensitivity were observed only when MgATP was provided
through the CK reaction, the results strongly suggest that
H2O2 mainly acts through an inhibition of the
myofibrillar CK and that this inhibition is responsible for
Ca2+ sensitivity changes. Similar results were obtained
after incubation with XO (30 IU/mL) and xanthine (100 µmol/L) (data
not shown).
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Effects of H2O2 on Tension Kinetics
and Stiffness
We have shown previously that inhibition of myofibrillar CK
changes the local ATP-to-ADP ratio and alters the mechanical
properties.28 Local deficiency in MgATP or more possibly
MgADP accumulation26 27 tends to inhibit crossbridge
detachment (or the transition from strong to weak binding states) and
thus to slow down the overall crossbridge cycling rate. Since a higher
number of crossbridges is attached at a given moment, the
Ca2+ sensitivity of tension development and the overall
amount of Ca2+ bound to the regulatory proteins are
increased.26 30 31 32
When an activated fiber is subjected to quick changes in
length, the immediate tension response to the length change is
determined by the number of attached crossbridges; the kinetics of the
return of tension to control level depends on the crossbridge cycling
rate.21 Quick length changes were thus applied to test
whether the ROS affect crossbridge kinetics and stiffness. Tension
responses to quick length changes were first determined in the relaxing
solution to assess passive mechanical properties. Responses in relaxing
solutions were then subtracted from responses in control activating
solution (pCa 4.5) to determine active mechanical properties. The
preparations were then successively bathed in solutions containing
increasing concentrations of H2O2 in the
presence of ADP and PCr. We observed that fibers studied in solutions
that did not contain a protector of SH groups (DTT) were less stable
and evidenced a time-dependent increase in the time constant of
tension recovery after stretches as well as a decrease in tension and
absolute stiffness. The rundown of the preparations was measured in a
parallel series of experiments, and the values obtained with
H2O2 were corrected accordingly. The results of
such an experiment are shown in Fig 7. Results obtained
on four different fibers showed that H2O2
increased the time constant of tension recovery in a dose-dependent
manner, ie, decreased the cycling rate of the crossbridges. These
changes were already significant at 0.5 mmol/L
H2O2, whereas stiffness normalized to
force started to increase for higher (1 to 2 mmol/L) concentrations. An
increase in this parameter indicates a decreased force
generated per crossbridge.
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The time constant of tension changes was also estimated before and after incubation of the fiber for 25 minutes with 2.5 mmol/L H2O2. In the presence of MgATP alone (solution D), the time constant of tension recovery was not altered by H2O2 incubation (37.0±2.0 versus 36.2±1.5 milliseconds in control, n=4). In the presence of PCr (solution F), the time constant of tension changes was dramatically increased from 19.1±1.6 to 49.5±3.3 milliseconds (n=5, P<.001) after incubation with H2O2.
Effects of H2O2 or X/XO on Myosin ATPase
and Myofibrillar CK–Specific Activities
The enzymatic activities of myofibrillar CK and myosin ATPase have
been measured on Triton-skinned isolated cardiac cells in
conditions similar to those of mechanical experiments with skinned
fibers. Isolated cardiac cells were incubated in 1 mL solution A for 25
minutes at 22°C in different conditions. Enzymatic activities were
measured for each sample before and after incubation.
Control myofibrillar CK activity was 1.27±0.03 IU/mg protein (n=26).
As shown in Fig 8A, CK was inhibited by incubation with
2.5 mmol/L H2O2; this inhibition was prevented
by 100 IU/mL CAT. Incubation with 10 µmol/L PMSF and 100 µmol/L
xanthine did not modify CK activity. XO (30 mIU/mL) decreased CK
activity in a xanthine concentration–dependent manner. This
inhibition was prevented by the addition of both SOD (400 IU/mL) and
CAT (100 IU/mL) as well as by the SH group reducing agent DTT (1
mmol/L).
|
Mg-ATPase activity of control cells was 32.8±1.6 mIU/mg protein (n=8)
and Ca-ATPase activity was 97.7±4.9 mIU/mg protein (n=8). Myosin
ATPase activities either in the absence or in the presence of
Ca2+ were unaffected by 25 minutes of incubation with X/XO
or H2O2 at 22°C (Fig 8B), thus confirming the
conclusions obtained from the mechanical experiments.
|
Discussion |
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|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is largely recognized that ROS may affect myocardial function. Numerous possible sites of action have been reported, and among them myofibrillar proteins have been involved. The results of the present study show the following: (1) The decrease in maximal force previously reported in experiments using the X/XO system could be attributed to a high contaminating proteolytic activity of commercial XO preparations. (2) The effects of ROS on rigor tension development, Ca2+ sensitivity, and rate constant of tension recovery were dependent on substrate and were not observed in the absence of PCr. (3) The main effect of ROS was a strong inhibition of myofibrillar CK without alterations of myosin ATPase. (4) The effects of H2O2 and XO were similar. (5) The effects were largely prevented by DTT. These results suggest that the main effect of ROS in myofibrils is the oxidation of the essential SH groups of CK, inducing an inactivation of the enzyme and a decrease in the local ATP-to-ADP ratio.
ROS and Tmax
In the present study, we attributed the fall in Tmax, observed
after application of XO on maximally Ca2+-activated
fibers, to the contaminating proteolytic activity of commercial XO
preparations. This conclusion was drawn after several observations: (1)
The fall in maximal Ca2+-activated tension could be
observed in the absence of substrate for XO and could not be prevented
by high activities of both CAT and SOD. (2) These effects were very
similar to those of trypsin, a well-known proteolytic enzyme. (3)
They could be antagonized by PMSF, an inhibitor of
proteases that did not interfere with the production of
superoxide by the X/XO system. In addition, commercial preparations of
XO do contain proteolytic activity (eg, see Sigma catalog 1995, p
1052).
Mammalian XO exists originally as a dehydrogenase form and is converted
to an oxidase form either irreversibly by proteolysis or reversibly by
SH oxidation of the protein molecule.25 The pretreatment
of XO with PMSF, which prevented tension decrease, did not interfere
with the production of O2-. We
could thus clearly conclude that the decrease in maximal force induced
by crude XO and described in skinned cardiac fibers (References 17 and
1817 18 and the present study) appeared to be the consequence of the
contaminating proteolytic activity of commercial XO preparations.
Inhibition of CK Is Responsible for the ROS-Induced Alterations of
Myofibrillar Function
Incubation with either X/XO (100 µmol/L per 30 mIU/mL) or 2.5
mmol/L H2O2 induced several alterations of
myofibrillar function, ie, an increase in resting tension at low ATP,
an increase in Ca2+ sensitivity, a decrease in Hill
coefficient, and a slight augmentation of maximal force. These effects
appeared to be directly related to ROS, since they could be antagonized
by CAT and SOD, enzymes catalyzing the degradation of the ROS. We found
that these alterations in myofibrillar function were highly dependent
on the pattern of energy supply. Withdrawal of PCr induced an increase
in Ca2+ sensitivity as well as a slight rise in resting
tension.28 However, incubation with the ROS did not
produce a further change in Ca2+ sensitivity. By contrast,
when ATP was generated through the phosphorylation of
ADP by myofibrillar-bound CK and PCr, strong increases in
Ca2+ sensitivity and in resting tension were observed.
Likewise, the dependence of rigor tension development as a function of
MgATP was not altered by either X/XO or H2O2 in
the absence of PCr but was greatly altered in its presence. The same
held true for the time constant of tension changes. Increases in
Ca2+ sensibility and in the time constant of tension
changes and the shift of the pMgATP-tension dependence to higher MgATP
concentrations are reminiscent of the effects brought about by a PCr
drop.28
Ca2+ sensitivity of tension development may be influenced
by a number of factors. Among them, the decreased rate of crossbridge
cycling due to substrate deficiency may induce an increase in resting
tension, an increase in Tmax and stiffness, and a shift toward lower
Ca2+ concentrations of the pCa-tension relationship
together with a decrease in cooperativity.28 30 This
effect can be observed when comparing the pCa-tension relationships
obtained with or without PCr (Fig 6 and Reference 2828 ) and can be
explained by the efficacy of bound CK to maintain a high local
ATP-to-ADP ratio. Under conditions of restricted exchange of adenine
nucleotides between the cytosol and the myofibrillar
compartment, bound CK can provide MgATP, remove MgADP, and buffer
proton production in the immediate environment of myosin ATPase
(for review, see Reference 2626 ). Inhibition of the CK function leads to
a slowing of the off rate of crossbridges and a prolonged duration of
the duty cycle of crossbridges.
These observations give evidence that the myofibrillar function is not altered directly through modifications of the regulatory proteins or myosin ATPase but indirectly through an inhibition of myofibrillar CK–inducing substrate deprivation of myosin ATPase and secondary slowing down of crossbridge kinetics. This was confirmed by the measurements of myofibrillar CK and myosin ATPase activities in our experimental conditions. Although incubation with both H2O2 and X/XO was able to dramatically decrease myofibrillar CK activity, neither myosin Mg-ATPase nor Ca2+-activated ATPase was affected.
Sensitivity of Myosin ATPase and Myofibrillar CK to
ROS
In the present experimental conditions, we did not obtain
evidence of alterations of myosin ATPase or regulatory proteins with
the ROS, whereas myofibrillar CK activity was greatly impaired. This is
in contrast with the fact that both enzymes have been shown to be
sensitive to ROS13 14 15 and that many SH groups are
present in myofibrils. The fact that no detectable alteration in
myofilament properties could be observed does not exclude the
occurrence of contractile protein alterations without clear functional
counterpart. In this respect, the increase in resting tension observed
after exposure to ROS in high Ca2+ (Reference 1818 and Fig 1
in the present study) could be explained in such a way. On the
other hand, a lower sensitivity of myosin ATPase compared with CK to
ROS could be involved. Indeed, Kaneko et al15 reported a
complete inhibition of myofibrillar CK by X/XO within a few minutes at
37°C with 0.5 mmol/L xanthine and 7.5 mIU/mL XO, whereas
others13 showed that a maximum of 60% inhibition of
myosin ATPase required 30 minutes at 30°C with 30 mIU/mL XO and 2
mmol/L xanthine. Indeed, prolonged exposure to ROS might reveal myosin
ATPase alterations, and the observations of rigor tension finally
exceeding Tmax after prolonged exposure to HOCl18 may be
explained by the combined inhibition of both CK and
Ca2+-activated myosin ATPase.
CK System in Cardiac Tissues
CK is an important cellular enzyme involved in energy transduction
in muscle cells. In addition to the traditional role of CK as a
spatiotemporal ATP/ADP buffer, it has been proposed that the location
of the CK isoenzymes in cardiac muscle facilitates the transduction of
high-energy phosphates throughout the cell and acts to
fine-tune the regulation of energy utilization and
production.33 34 In myofibrils, CK is an integral
part of the M line (for review, see Reference 3535 ) and is also
distributed across the entire filament.36 There is a high
activity of myofibrillar CK (1 to 2 IU/mg protein). This
myofibrillar-bound CK can induce a specific kinetic enhancement of
the myofibrillar ATPase37 and
rephosphorylate enough MgADP produced by the myosin
ATPase reaction to ensure optimal contractile capacities as well as
normal Ca2+ sensitivity and crossbridge cycling rate in the
absence of MgATP and thus can meet the contractile protein energy
requirements.26 28 35 On the other hand,
deprivation of PCr, even in the presence of ATP, decreases
the rate of cycling of crossbridges, increases the Ca2+
sensitivity, and increases the susceptibility of rigor tension
development to a decrease in MgATP. We have recently shown that the
inhibition of myofibrillar function following the sharp decrease in PCr
seen during ischemia or hypoxia seems to be responsible
for rigor tension development, which is the basis of the
ischemic contracture.27
The CK isoenzymes contain easily oxidized SH groups in the active site, which are required for the enzymatic activity.38 Inhibition of mitochondrial as well as cytosolic forms of CK by the ROS has been described previously.13 15 39 40 Thus, alteration of myofibrillar CK by the ROS may be one of the mechanisms involved in the paradoxical reduction of contractile activity at reperfusion despite complete and fast recovery of PCr content.41 Indeed, altered myofibrillar CK activity was shown upon reperfusion in open-chest dogs subjected to 15 minutes of left anterior descending coronary artery occlusion.42 The failure to observe altered myofibrillar function and myofibrillar CK in isolated rat hearts after ischemia and reperfusion43 44 could be due either to low ROS generation in isolated heart preparations or to the presence of radical scavengers or reducing agents in these in vitro experiments.
ROS and Myofibrillar Function
In our experiments, the X/XO system and
H2O2 produced similar effects on myofibrillar
properties and CK. The production of free radicals has been
measured in both conditions, in the relaxing solution and in the
absence of skinned fibers. EPR spectra confirmed the generation of
superoxide radicals by the X/XO system45 but not hydroxyl
radicals. H2O2 did not generate any significant
hydroxyl radical or superoxide, as previously reported.46
In fact, H2O2 can be reduced to hydroxyl
radicals in the presence of iron, which facilitates the Fenton
reaction. In the presence of fibers, it could be possible that
endogenous iron catalyzes the reaction and that hydroxyl
radicals are produced with the two free radical–generating
systems. However, since desferrioxamine, an iron chelator, was without
effect, it is reasonable to consider that hydroxyl radicals were not
importantly involved and that this was merely due to the presence in
the solutions of EGTA, another metal chelator. In experiments performed
in intact papillary muscles, Schrier and Hess10 reported
that the main negative inotropic radical species produced by X/XO was
the superoxide anion and that H2O2 itself was
not involved. H2O2 toxicity could be due to
direct effects,46 but the formation of derived-toxic
substances could also be involved.
The exact mode of action of ROS is not well understood. Inhibitory effects of X/XO, HOCl, and H2O2 on myosin ATPase and CK were shown to be prevented by thiol-reducing agents such as DTT,13 14 15 suggesting that oxidation of SH groups was the main effect of ROS. DTT by itself does not decrease the high sensitivity of myofibrils to ATP in the presence of PCr, since pMgATP50 values are identical with or without DTT (values of 5.07 in Reference 27 and 527 5 .03 in the present study). In our hands, DTT did not completely reverse the effects of ROS on rigor tension development and was without effect on HOCl-induced mechanical alteration.18 These observations are not entirely consistent with a simple oxidation of thiol groups and require further analysis.
Relevance to In Vivo Conditions
The high effective concentrations of ROS in the present study
require further discussion. In the present study, mechanical
modifications were observed only for relatively high ROS concentrations
(30 mIU/mL XO or 2.5 mmol/L H2O2), whereas
lower concentrations of H2O2 (100 µmol/L) or
XO/X [(3 mIU/mL)/(30 µmol/L)] were without noticeable effects. In
the study of MacFarlane and Miller,18 prolonged
incubations with H2O2 concentrations up to 10
mmol/L only marginally increased resting tension, Tmax, and
Ca2+ sensitivity. On the other hand, Banerjee et
al41 and Suzuki et al14 observed an
inhibition of CK by 100 µmol/L H2O2. Limited
intracellular diffusion of these compounds seems to be excluded.
H2O2 is a small molecule, and fibers skinned
with Triton X-100 are permeable to ions, substrates, and enzymes.
Moreover, steady state effects were analyzed after prolonged
times of incubation. Similarly, superoxide production with XO
should take place both inside and outside the fibers. It is highly
possible that some compounds present in the solutions used for
skinned fiber experiments may have a protective effect against
H2O2 or other ROS action, thus explaining the
discrepancies in the dose-response relationship of myofibrillar CK
to H2O2. In addition, temperature differences
can also be involved. Our experiments were performed at 22°C, a
temperature that is known to increase the stability of the preparations
and that allows comparisons with other studies. Increasing the
temperature to more physiological values will
undoubtedly potentiate the ROS production and/or effects.
Indeed, Banerjee et al reported a 50% inhibition of CK activity for 30
µmol/L H2O2 after incubation for 15 minutes
at 37°C. Similarly, the higher sensitivity of myosin ATPase and CK to
ROS observed by Suzuki et al13 and Kaneko et
al15 may well be explained by the higher temperature used
in these studies. In this respect, an ED50 of 30 µmol/L
H2O2 for CK is in the plausible range of
physiologically relevant
H2O2 concentrations at
reperfusion.41 A similar involvement of temperature may
also apply to the X/XO system. Changes in a variety of oxidative
indexes in isolated perfused rat heart tissue subjected to direct
infusion of 10 mIU/mL XO for 10 minutes at 36°C were shown to be
similar in magnitude to those induced by as little as 10 minutes of
hypoxia.47 Although comparisons are difficult to
make, in the conditions of the present study, the intensity of free
radical generation with 30 IU/mL XO and 100 µmol/L xanthine was only
two to three times higher than that observed after reperfusion of an
isolated ischemic heart.4 Moreover, it should be
kept in mind that in skinned fiber experiments, because of the presence
of EGTA, hydroxyl radicals (the most reactive of the ROS) were not
generated. However, Khalid and Ashraf48 recently provided
evidence that OH· formation by isolated cells during cycles of
anoxia/reoxygenation was not the major ROS inducing
cell damage and enzyme leakage. Other in vivo sources of free radicals
and ROS may also be involved, such as the very reactive hypochlorous
acid that was shown to induce similar effects on skinned fibers at very
low concentrations.18 Taken together, these observations
suggest that inhibition of myofibrillar CK may occur in vivo under
conditions of ischemia/reoxygenation.
ROS Inhibition of CK and Cardiac Pathophysiology
A number of studies indicate a possible role of ROS in the
prolonged depression of the mechanical function following a brief
period of ischemia, known as myocardial
stunning.49 After restoration of the coronary
flow, the cellular ATP pool remains depleted for several days, whereas
PCr content often quickly returns back to preischemic
levels or even higher. During myocardial ischemia, a series of
metabolic events promotes the univalent reduction of
molecular oxygen and initiates free radical chain reactions. During
ischemia or hypoxia and
reoxygenation, involvement of free radical
generation has been documented. Formation of superoxide radicals and
H2O2 has been detected during
recirculation.4 41 The proteolytic conversion of xanthine
dehydrogenase to XO during Ca2+ overload by the activation
of Ca2+-sensitive proteases has been proposed as one of the
possible sources of free radicals (see Reference 11 for review).
However, even though XO activity is high in rat myocardium,
it appears to be absent in human myocardium.50
The involvement of XO in reperfusion injury is an attractive
hypothesis, but it is still a matter of debate.25
It is well documented that reperfusion after moderate ischemia is accompanied by a rapid recovery of PCr to supranormal levels (PCr overshoot), whereas contractile activity recovers slowly, suggesting an impairment in PCr utilization.41 51 52 53 During prolonged mild to moderate ischemia in anesthetized pigs, the return of PCr levels to control values after the first minutes of initial decrease, despite ATP concentrations of <50% of control values, suggested a limitation of the cells' ability to produce ATP by use of PCr.54 The decrease in total CK activity at reperfusion has been correlated with the generation of H2O2.41 Thus, paradoxically, elevated levels of PCr and depressed myocardial function during myocardial stunning were suggested to result from specific inhibition of cytosolic and myofibrillar CK by ROS.40 41 The present results are also consistent with this interpretation. However, apart from the fact that myocardial stunning is of multifactorial origin, this explanation requires that mitochondrial CK not be inhibited by free radicals, an assumption that is difficult to reconcile with electron flow through the respiratory chain being an important source of oxygen radicals.55 In addition, reductions in both cytosolic and mitochondrial forms of CK activity have been reported to occur in hearts subjected to ischemia and reperfusion.40 42 56 Such inhibition may induce serious alterations of cardiac function. Recently, using iodoacetamide to inhibit CK in isolated heart, Hamman et al57 concluded that CK activity was essential for the expression of the full dynamic range of myocardial performance and that myofibrillar CK inhibition could be the cause of altered kinetics of the myofibrillar ATPase and could contribute to a substantial reduction of contractile reserve.
Decreased bound as well as cytosolic CK activities induced by the ROS after ischemia and reperfusion could be of importance for the regulation of energy fluxes and the integration of utilization and production of high-energy phosphate compounds in the myocardium. The present study brings evidence that myofibrillar CK is the first and main target of the ROS in the myofibrillar compartment and that alteration of PCr utilization at the myofibrillar side of the PCr shuttle may participate in the damaging effects of ROS in cardiac cells. This effect may be partly responsible for the poor and slow recovery of mechanical function after ischemia and reperfusion.
|
Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported in part by grants from the Association Française contre les Myopathies, Fondation pour la Recherche Médicale, and Fondation de France. Dr Ventura-Clapier was supported by the Centre National de la Recherche Scientifique. The authors wish to acknowledge F. Lefèbvre for skillful technical assistance, P. Lechêne for engineering assistance, and R. Fischmeister for continuous support.
Received October 30, 1995; accepted March 5, 1996.
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