This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gao, W. D. Articles by Marban, E. Search for Related Content PubMed PubMed Citation Articles by Gao, W. D. Articles by Marban, E. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB CALCIUM COMPOUNDS CALCIUM, ELEMENTAL HYDROGEN PEROXIDE

(Circulation. 1996;94:2597-2604.)
© 1996 American Heart Association, Inc.

Articles

Selective Effects of Oxygen Free Radicals on Excitation-Contraction Coupling in Ventricular Muscle

Implications for the Mechanism of Stunned Myocardium

Wei Dong Gao, MD, PhD; Yongge Liu, PhD; Eduardo Marban, MD, PhD

the Section of Molecular and Cellular Cardiology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Md.

Correspondence to Eduardo Marban, MD, PhD, Room 844, Ross Bldg, Johns Hopkins University School of Medicine, 720 Rutland Ave, Baltimore, MD 21205. E-mail marban@welchlink.welch.jhu.edu.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background Oxygen free radicals (OFRs) have been implicated in the pathogenesis of myocardial stunning, but the precise mechanism by which OFRs foster stunning remains unclear. We investigated the pathophysiology of the contractile dysfunction that occurs after direct exposure of OFRs to cardiac muscle and compared the results with the pathophysiology of stunned myocardium.

Methods and Results Trabeculae from the right ventricles of rat hearts were loaded iontophoretically with fura-2 to determine [Ca²⁺]_i. Steady-state force-[Ca²⁺]_i relations were obtained by rapid electrical stimulation in the presence of ryanodine. Two exogenous OFR-generating systems were used: H₂O₂+Fe³⁺-nitrilotriacetic acid (H₂O₂+Fe³⁺) to produce hydroxyl radical, and xanthine oxidase+purine (XO+P) to produce superoxide. In muscles exposed to H₂O₂+Fe³⁺ for 10 minutes, both twitch force and Ca²⁺ transients were decreased (eg, in 1.5 mmol/L external [Ca²⁺], force decreased from 41±7 to 23±4 mN/mm², P<.05, and Ca²⁺ transient amplitude from 0.96±0.09 to 0.70±0.05 µmol/L, P<.05). Maximal Ca²⁺-activated force (F_max) decreased slightly, from 103±5 to 80±12 mN/mm² (P=NS). Neither the [Ca²⁺]_i required to achieve 50% of F_max (Ca₅₀) nor the Hill coefficient was changed. In muscles exposed to XO+P for 20 minutes, twitch force was reduced (in 1.5 mmol/L external [Ca²⁺]) from 50±9 to 39±8 mN/mm² (P<.05). Ca²⁺ transients, on the other hand, were not affected. F_max decreased insignificantly from 100±16 to 81±14 mN/mm². Ca₅₀ increased from 0.71±0.06 to 1.07±0.07 µmol/L (P<.05), with no change in the Hill coefficient.

Conclusions These results indicate that exposure to the H₂O₂+Fe³⁺ free radical–generating system reduces activator Ca²⁺ availability, whereas XO+P decreases the Ca²⁺ sensitivity of the myofilaments. Exogenously generated OFRs, particularly those produced by XO+P, mimic the effects of myocardial stunning on cardiac excitation-contraction coupling.

Key Words: free radicals • calcium • myocardial contraction

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Oxygen free radicals have been the focus of intensive investigation as potential mediators of myocardial ischemia-reperfusion injury. Several lines of evidence implicate OFRs in the reversible form of injury known as "stunning."¹ OFRs are generated in reperfused myocardium,^{2 3 4} and administration of OFR scavengers attenuates stunning.^{5 6 7 8 9} Nevertheless, the mechanism by which OFRs may produce stunning is poorly understood. OFRs have widespread effects on the myocardium,¹ including inhibition of myofibrillar creatine kinase activity¹⁰ and a direct depressant effect on the myofilaments evident in skinned cardiac muscles exposed to OFR-generating systems.^{11 12} Since myocardial stunning is characterized by a decreased myofilament Ca²⁺ responsiveness,^{13 14} the latter studies provide an explicit mechanism for OFR-induced stunning. However, the production of the primary OFRs, such as ·O₂^-, in reperfused myocardium lasts for only a few minutes.^{2 3 4} Thus, the continuous presence of OFRs in previous studies and the direct exposure to the myofibrillar apparatus make it difficult to extrapolate the observed effects to myocardial stunning.

In the present study, we evaluated the mechanisms underlying the decreased contractile performance in rat cardiac trabeculae transiently exposed to exogenously generated OFRs. Two different OFR-generating systems were compared: H₂O₂+Fe³⁺ and XO+P. Our results demonstrate that many phenotypic features of stunning were reproduced by exposure to XO+P and less so by exposure to H₂O₂+Fe³⁺. The differential effects of different OFR-generating systems on excitation-contraction coupling have important implications for the mechanism of myocardial oxidant injury.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Preparation
Rats (LBN-F1 brown rats, 200 to 250 g, Harlan, Indianapolis, Ind) were anesthetized by intra-abdominal injection of sodium pentobarbital (0.1 to 0.2 mL), and the hearts were rapidly excised via a midsternal thoracotomy. Trabeculae from the right ventricles were quickly dissected. The dimensions of the trabeculae (in mm) were 2.38±0.60 long, 0.20±0.10 wide, and 0.11±0.03 thick (mean±SD, n=25). After dissection, the trabeculae were mounted between a force transducer and a micromanipulator in a perfusion bath. The trabeculae were superfused with KH buffer equilibrated with 95% O₂/5% CO₂. The KH buffer was composed of (in mmol/L) Na⁺ 142, K⁺ 5, Mg²⁺ 1.2, Cl^- 127.4, PO₄^- 2, HCO₃^- 20, and CaCl₂ 0.5, pH 7.35 to 7.4. The perfusion rate was 10 mL/min, and the preparations were stimulated at 0.5 Hz. All experiments were performed at room temperature (20°C to 22°C) to parallel previous work on stunned myocardium in this experimental system.¹⁴ Force and sarcomere length were measured as described previously.¹⁴ Diastolic sarcomere length was set to 2.2 to 2.3 µm and was monitored throughout the experiments. Sarcomere length did not change before and after treatment with OFR-generating systems.

Measurement of Intracellular [Ca²⁺] With Fura-2
[Ca²⁺]_i was measured by the method described previously.^{14 15} Fura-2 potassium salt was microinjected iontophoretically into one cell and allowed to spread throughout the whole muscle (via gap junctions). The loading did not affect force development.

[Ca²⁺]_i was determined by measurement of the epifluorescence of fura-2 in the cells excited by ultraviolet light at 380 and 340 nm.^{14 15} The fluorescent light was collected at 510 nm by a photomultiplier tube (R1527, Hamamatsu). The output of the photomultiplier tube was filtered at 100 Hz, collected by an analog-to-digital converter, and stored in the computer for later analysis.

Intracellular [Ca²⁺] was given by the following equation (after subtraction of the autofluorescence of the muscle):

(E1)

where R is the observed ratio of fluorescence (340/380), K'_d is the apparent dissociation constant, R_max is the ratio of 340/380 nm at saturating [Ca²⁺], and R_min is the ratio of 340/380 nm at zero [Ca²⁺]. The values for K'_d, R_max, and R_min were 2.95 µmol/L, 9.55, and 0.50, respectively, as determined by in vivo calibrations in trabeculae.¹⁴ When K'_d is corrected for an instrument-specific optical factor (S_f2/S_b2 in Grynkiewicz et al¹⁶ ), the true K_d value for Ca²⁺ binding to fura-2 is estimated to be 280 nmol/L.

External OFR Generation
In this study, OFRs were produced in two ways. (1) The H₂O₂+Fe³⁺-NTA system generates the very toxic hydroxyl radical (·OH), via the Fenton reactions, as

(E2)

(E3)

The sum of Equations 2 and 3 is

(E4)

In our experiments, 1 mmol/L H₂O₂ and 0.1 mmol/L of the ferric iron chelator Fe³⁺-NTA were used. The amount of ·OH generated under these conditions was roughly comparable to that produced during ischemia-reperfusion.^{4 17} (2) The XO+P system produces primarily the superoxide radical (·O₂^-), via the following reaction:

(E5)

(E6)

The reactions are catalyzed by xanthine oxidase. Concentrations of 3.0 mmol/L purine and 0.05 U/mL XO were used.

Experimental Protocol
After loading of fura-2, trabeculae were divided into one of four groups: For group 1 (n=8), the H₂O₂+Fe³⁺ group, 0.1 mmol/L Fe³⁺-NTA was added to the superfusate. After 2 to 3 minutes, H₂O₂ (1 mmol/L) was also added to the solution. After 10 minutes of H₂O₂+Fe³⁺-NTA, the perfusion was switched back to normal KH solution. For group 2 (n=6), the control group for the H₂O₂+Fe³⁺ system, trabeculae were superfused with KH solution containing 0.1 mmol/L Fe³⁺-NTA alone for 10 minutes and then continuously superfused with normal KH solution again. For group 3 (n=7), the XO+P group, trabeculae were initially superfused with normal KH solution, and then purine (3.0 mmol/L) was added to the solution. After 3 minutes, XO (0.05 U/mL) was added. After 20 minutes of XO+P, the perfusion was switched back to normal KH solution. For group 4 (n=4), the control group for XO+P, the protocol was the same as group 3 except that inactivated XO (incubated in boiling water for 5 minutes) was used. The concentrations of the various components in the free radical–generating systems were based on published values,^{4 17 18 19} and the durations of exposure were chosen so as to produce a decrease in twitch contraction of 40% to 50% at steady state during washout, on the basis of preliminary experiments.

After measurement of twitch contractions and the underlying Ca²⁺ transients at external [Ca²⁺] from 0.5 to 2.0 mmol/L, all muscles were treated with 1 µmol/L ryanodine for 15 to 20 minutes. Various steady-state activations were obtained by stimulating the muscles at 10 Hz in different external [Ca²⁺]. Steady-state force-[Ca²⁺]_i relations were fit with a function of the following form (Hill equation): F=F_max[Ca²⁺]ⁿ/(Ca₅₀ⁿ+[Ca²⁺]ⁿ), where F_max is the maximal Ca²⁺-activated force, Ca₅₀ is the [Ca²⁺]_i required for 50% of maximal activation, and n is the Hill coefficient.¹⁴

Statistical Analysis
Paired Student's t test, linear regression analysis, one-way ANOVA, or multivariate ANOVA was used for statistical analysis of the data as indicated.^{20 21} A value of P<.05 was considered to indicate significant differences between groups. Unless otherwise indicated, pooled data are expressed as mean±SEM.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Force Development After Brief Exposure to H₂O₂+Fe³⁺ in Trabeculae
Fig 1A shows a continuous record of force development in a trabecula before, during, and after exposure to H₂O₂+Fe³⁺ (1.0 mmol/L external [Ca²⁺]). Addition of Fe³⁺ alone did not change force development. After the addition of H₂O₂, force first decreased gradually. At about 9 minutes, force started decreasing more abruptly, and diastolic force began to rise. On return to normal KH solution, diastolic force continued to increase before eventually decreasing. Force development started to recover as well. In about 25 to 30 minutes, force reached a new steady state at 45% of the force before exposure. Despite the transient contracture during exposure to the free radical–generating system, there was no maintained change in diastolic force after washout. Fig 1B shows individual twitch contractions (top) before and after the exposure and the corresponding Ca²⁺ transients (bottom) of the same trabecula. It is clear that after exposure, both force and Ca²⁺ transients were decreased. When pooled data of peak force and peak [Ca²⁺]_i were plotted against each other (Fig 1C), the H₂O₂+Fe³⁺ data (solid squares) fell along the same general relationship as the data before exposure (open squares).

View larger version (14K): [in this window] [in a new window]   Figure 1. Effects of hydroxyl radical generation on force and [Ca2+]i. A, Raw record of force before, during, and after exposure to H2O2+Fe3+ in a trabecula. Note that addition of Fe3+ alone did not affect force. B, Typical record of force and Ca2+ transients before (left) and after (right) 20 minutes of washout of H2O2+Fe3+ in 1.0 mmol/L external [Ca2+] from trabecula shown in A. Before exposure, force was 46.0 mN/mm2 and peak [Ca2+]i was 0.94 µmol/L. Both force and [Ca2+]i were concomitantly decreased and abbreviated after treatment with H2O2+Fe3+ (force, 24.8 mN/mm2; systolic [Ca2+]i, 0.53 µmol/L). C, Pooled data of peak force and peak [Ca2+]i from all muscles (n=8) before () and after () exposure to H2O2+Fe3+. Data were fitted with linear functions. Line indicates best fit for all data (slope, 41 mN/mm2·µmol/L).

Fig 2 summarizes the data of [Ca²⁺]_i (left) and force (right) at varied external [Ca²⁺] (0.5 to 2.0 mmol/L) before and after exposure to the free radical–generating system. Both peak systolic [Ca²⁺]_i and peak systolic force decreased in parallel after exposure to H₂O₂+Fe³⁺ as external [Ca²⁺] increased. There were no significant changes in diastolic [Ca²⁺]_i and force.

View larger version (19K): [in this window] [in a new window]   Figure 2. Pooled data of systolic and diastolic [Ca2+]i (left) and systolic and diastolic force (right) at varied external [Ca2+] before (open bars) and after (solid bars) treatment with H2O2+Fe3+ in rat trabeculae at varied [Ca2+]. *P<.05 by paired t test; n=8.

To characterize excitation-contraction coupling during twitch contractions more fully, the Table quantifies the changes in the time course of Ca²⁺ transients and force before and after H₂O₂+Fe³⁺ exposure at varied external [Ca²⁺]. Time to peak Ca²⁺ transients increased significantly (P<.05, multivariate ANOVA) at all external [Ca²⁺]. Time to peak force increased insignificantly. Time from peak to 50% decay of Ca²⁺ transients did not change, but relaxation of force was abbreviated (P<.05, multivariate ANOVA). This acceleration of relaxation may simply be due to the decreased twitch force, which influences the rate of relaxation, as described previously.²² Taken together, the data in Figs 1 and 2 and the Table suggest that the decreased force was caused simply by a decreased amplitude of Ca²⁺ transients after H₂O₂+Fe³⁺ exposure.

View this table: [in this window] [in a new window]   Table 1. Effects of H2O2+Fe3+ and XO+P Systems on the Kinetics of Ca2+ Transients and Twitch Force

Effects of Brief Exposure to H₂O₂+Fe³⁺ on Steady-State Force-[Ca²⁺]_i Relation in Rat Trabeculae
As a further check on the idea that the decreased force was caused solely by reduced Ca²⁺ transients, we investigated whether the steady-state Ca²⁺ responsiveness of the myofilaments was affected. Since agents that interfere with SR function may potentially affect the outcome of exposure to H₂O₂+Fe³⁺, these experiments were performed in an unpaired fashion: trabeculae were divided into two experimental groups (see "Methods," groups 1 and 2). Fig 3 shows the force-[Ca²⁺]_i relations from pooled data for these two groups. The data were normalized to their respective maximal values; the absolute values of F_max are shown in the inset. We found that F_max (103±5 versus 80±12 mN/mm², P=NS), Ca₅₀ (0.60±0.07 versus 0.69±0.07 µmol/L, P=NS), and the Hill coefficient (6.76±0.92 versus 6.78±0.82, P=NS) did not change significantly. The absence of any significant effect confirms the idea that a deficiency of activator Ca²⁺ is the primary factor that underlies the depressant effect of H₂O₂+Fe³⁺.

View larger version (19K): [in this window] [in a new window]   Figure 3. Steady-state force-[Ca2+]i relations of rat trabeculae without () (n=6) and after () (n=7) treatment with H2O2+Fe3+. Data within various bins of [Ca2+]i were pooled and represented for clarity. Absolute values of Fmax are plotted in inset. All submaximal forces were normalized with respect to each group's own Fmax. Fits represent Hill function based on mean values of Fmax, Hill coefficient (n), and Ca50.

Force Development in Trabeculae After Exposure to XO+P
The results above demonstrate the effects of brief exposure to H₂O₂+Fe³⁺ on contractile performance. We next studied the effects of exposure to XO+P. In this series of experiments, trabeculae were exposed to XO+P for 20 minutes before they were returned to normal KH solution. Fig 4 shows the ensuing changes of force and Ca²⁺ transients. Fig 4A shows a typical recording of force development before, during, and after XO+P exposure in 1.0 mmol/L external [Ca²⁺]. Addition of purine alone increased force slightly, but this was not seen consistently in all muscles. On addition of XO, there was an initial increase in systolic force, followed by a progressive decrease over 20 minutes to a low level. Diastolic force rose slightly and remained elevated throughout exposure to XO+P. When trabeculae were returned to normal KH buffer, diastolic force returned to the control level, whereas systolic force remained depressed at 30 minutes. Fig 4B shows representative individual twitch contractions and the corresponding Ca²⁺ transients before and after the exposure. Systolic force was decreased by about 50%, but in contrast to the depressant effect of H₂O₂+Fe³⁺, Ca²⁺ transients increased. Pooled data for peak force and peak [Ca²⁺]_i are plotted in Fig 4C. The results before and after exposure are clustered in two distinct populations of data points with different slopes when fitted with linear functions (46±7 versus 27±7 mN/mm²·µmol/L, P<.05).

View larger version (15K): [in this window] [in a new window]   Figure 4. Effects of superoxide generation on force and [Ca2+]i. A, Raw record of force before, during, and after exposure to XO+P in a trabecula. Addition of purine alone increased force slightly. B, Typical traces of force and [Ca2+]i transients before exposure (left) and after 30 minutes of washout (right) at 1.0 mmol/L external [Ca2+] from trabecula shown in A. Before exposure, force was 40.0 mN/mm2 and systolic [Ca2+]i was 1.12 µmol/L. Note that force was reduced, but [Ca2+]i increased after treatment with XO+P (force, 20.7 mN/mm2; systolic [Ca2+]i, 1.37 µmol/L). C, Pooled data of peak force and peak [Ca2+]i from all muscles (n=7) before () and after () exposure to XO+P. Data were fitted with linear functions. For control muscles, slope is 46.4 mN/mm2·µmol/L, and for XO+P-treated, 26.7 mN/mm2·µmol/L.

Fig 5 summarizes the changes in force and Ca²⁺ transients over a range of external [Ca²⁺]. After XO+P exposure, systolic [Ca²⁺]_i either remained unchanged or was increased at various external [Ca²⁺], whereas systolic force decreased significantly (P<.05, paired t test). Despite modest but significant increases in diastolic [Ca²⁺]_i (P<.05, paired t test), diastolic force did not change as external [Ca²⁺] increased. There was no change in the time course of Ca²⁺ transients or force after treatment with XO+P (Table).

View larger version (19K): [in this window] [in a new window]   Figure 5. Pooled data of systolic and diastolic [Ca2+]i (left) and systolic and diastolic force (right) at varied external [Ca2+] before (open bars) and after (solid bars) treatment with XO+P in rat trabeculae at varied [Ca2+]. *P<.05 by paired t test; n=7.

Effects of XO+P Exposure on Steady-State Force-[Ca²⁺]_i Relation
To measure Ca²⁺ responsiveness directly, the muscles were tetanized in the presence of ryanodine. Fig 6 shows the pooled force-[Ca²⁺]_i relations from control and XO+P trabeculae. As in Fig 3, the data were normalized to their respective maximal values, and the absolute values of F_max are shown in the inset. There was a rightward shift of the force-[Ca²⁺]_i relations after exposure to XO+P, with Ca₅₀ increasing significantly (0.71±0.06 versus 1.07±0.07 µmol/L, P<.05). There were, however, no significant changes in F_max (100±16 versus 80.9±14 mN/mm²) or Hill coefficients (5.57±0.90 versus 5.04±0.48).

View larger version (20K): [in this window] [in a new window]   Figure 6. Steady-state force-[Ca2+]i relations of rat trabeculae without () (n=4) and after () exposure to XO+P (n=7). Data within various bins of [Ca2+]i were pooled for clarity. Absolute values of Fmax are plotted in inset. All submaximal forces were normalized with respect to each Fmax. Fits represent Hill function based on mean values of Fmax, Hill coefficient (n), and Ca50.

Differential Effects of the Two OFR-Generating Systems on [Ca²⁺]_i
Stunning and its associated changes in myofilament Ca²⁺ responsiveness have previously been linked to calcium overload during early reperfusion.²³ Indeed, the stunned phenotype is reproduced when hearts have been subjected to transient calcium overload in the absence of ischemia.^{24 25} Thus, it is logical to wonder whether differences in [Ca²⁺]_i during exposure to XO+P versus H₂O₂+Fe³⁺ underlie the differential consequences of the two OFR-generating systems.

Fig 7 contrasts the changes in [Ca²⁺]_i during the two protocols. Exposure to XO+P (Fig 7A) leads to a significant increase in diastolic [Ca²⁺]_i by 10 minutes, which persists even after washout of the enzyme. Systolic [Ca²⁺]_i falls modestly during the exposure but recovers immediately on washout. In contrast, Fig 7B shows that the changes of [Ca²⁺]_i are qualitatively different with H₂O₂+Fe³⁺: diastolic [Ca²⁺]_i does not increase at all, and the decrease in systolic [Ca²⁺]_i is more profound than with XO+P. An increase of diastolic [Ca²⁺]_i is the hallmark of calcium overload.²⁶ Thus, the finding that diastolic [Ca²⁺]_i increases with XO+P but not H₂O₂+Fe³⁺ is consistent with the idea that Ca²⁺ is the final common mediator of stunning.²³

View larger version (11K): [in this window] [in a new window]   Figure 7. Changes of systolic (solid symbols) and diastolic (open symbols) [Ca2+]i before, during, and after exposure to XO+P (A, n=7) or H2O2+Fe3+ (B, n=8). All data were obtained at an extracellular [Ca2+] of 1.0 mmol/L. *P<.05 by paired t test vs before exposure.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Mechanisms of Radical-Mediated Cardiac Injury
The effects of OFRs on cardiac muscle have been extensively investigated. Contractile force and the rate of force development in papillary muscles are known to be decreased after 60 minutes of exposure to the XO+P free radical–generating system.¹⁹ Significant structural alterations have also been noted in interventricular septal preparations subjected to such a free radical–generating system.¹⁸ Specific cellular targets include the SR Ca²⁺ pump,²⁷ the sarcolemmal Ca²⁺ pump,²⁸ the sarcolemmal Na⁺,K⁺-ATPase,²⁹ myofibrillar ATPase,³⁰ and myofibril-associated creatine kinase.¹⁰ Electrophysiological abnormalities have also been observed in hearts and in cardiac cells exposed to OFR-generating systems. Action potential duration was abbreviated because of activation of ATP-sensitive K⁺ channels.³¹ Ca²⁺ current was found to be attenuated,^{31 32} whereas Na⁺/Ca²⁺ exchange was stimulated.³³ Recent experimental evidence suggests that many of the electrophysiological and functional abnormalities may be related to inhibition of energy metabolism, particularly glycolysis.^{17 31} Given their wide range of deleterious effects on cells, it is not surprising that OFRs decrease force development and eventually kill the cells.

Exposure to generating systems that produce either ·OH or ·O₂^- has generally been reported to cause identical types of injuries, differing only in the degree of damage. However, many of the above-mentioned results were obtained with continuous OFR generation for >60 minutes, producing damage that may have been sufficiently advanced as to mask the intermediary pathways from which those injuries arose. More recent studies have indicated that the effects of ·OH and ·O₂^- on cell function can be quite different. In vascular smooth muscle cells, only ·O₂^- was able to increase the activity of mitogen-activated protein kinase, a key factor in cell proliferation, even though both ·OH and ·O₂^- stimulated cell growth.³⁴ In skinned cardiac fibers, contraction was decreased significantly after 5 to 15 minutes of exposure to 4 mmol/L xanthine+XO, whereas such an effect was seen only after 35 minutes of exposure to 0.4 mmol/L FeCl₂+H₂O_2, despite similar or higher production of free radicals in the latter system.¹² In isolated rat myocytes, reoxygenation after 15 minutes of anoxia induced ·OH production, which was prevented by deferoxamine; however, LDH release was not attenuated by deferoxamine but rather by diphenylphenylenediamine, which retarded the production of malondialdehyde.³⁵ This study was interpreted as showing that free radicals other than ·OH caused most of the injury.

Differential Effects of Exposure of H₂O₂+Fe³⁺ and XO+P on Contractile Function
Our results reveal distinct differences in the effects of the two free radical–generating systems on cardiac trabeculae. The differences were first evident during exposure to the OFR-generating systems (Fig 7): XO+P increased diastolic [Ca²⁺]_i, but H₂O₂+Fe³⁺ did not. The aftereffects of the two OFR-generating systems also diverged. Although both decreased twitch force, they differed strikingly in their effects on Ca²⁺ transients and Ca₅₀: H₂O₂+Fe³⁺ reduced Ca²⁺ transient amplitude but did not change the Ca₅₀ of the force-[Ca²⁺]_i relation; in contrast, XO+P resulted in a slight increase in Ca²⁺ transients and a rightward shift of the force-[Ca²⁺]_i relation. These results indicate that, whereas the lasting effect of H₂O₂+Fe³⁺ was on [Ca²⁺]_i regulation, the primary aftereffect of XO+P was on the myofilaments.

The present study demonstrates that the two OFR-generating systems do not share a common mechanism in their depressant effects on myocardial contraction. H₂O₂+Fe³⁺ treatment decreased Ca²⁺ transients, thus decreasing the availability of activator Ca²⁺. H₂O₂+Fe³⁺ is known to generate hydroxyl radical (see above). Although we did not measure Ca²⁺ release from the SR directly, the target of ·OH may well have involved the SR Ca²⁺-releasing process, as evidenced by the delayed time to peak Ca²⁺ transients (Table) and the unchanged steady-state force-[Ca²⁺]_i relations after exposure to H₂O₂+Fe³⁺ (Fig 3). Diminution of SR Ca²⁺ release may reflect decreased influx of Ca²⁺ through sarcolemmal channels (ie, decreased amount of triggering Ca²⁺),³⁶ less Ca²⁺ release (in the presence of normal triggering amounts of Ca²⁺), or both. XO+P generates superoxide radical primarily, with subsequent production of ·OH depending on the presence of Fe³⁺ and the activities of superoxide dismutase and catalase. The XO+P system exerts its negative inotropic effect by acting on the myofilaments, since Ca²⁺ transients were not inhibited, but the Ca²⁺ sensitivity of the contractile proteins was decreased after treatment with XO+P (Figs 4 and 6).

The unique action of the H₂O₂+Fe³⁺ system on Ca²⁺ handling suggests that structures directly involved in Ca²⁺ release (eg, sarcolemmal Ca²⁺ channels and SR Ca²⁺ release channels) may be especially susceptible to ·OH and that such effects persist even after the removal of ·OH. It has been shown previously that Ca²⁺ current is reduced in the presence of OFRs.^{31 32} Studies of the effects of OFRs on the SR have shown degradation of the release channel³⁷ and impairment of Ca²⁺ release from the SR in saponin-treated skinned muscles,³⁸ but the pathophysiological implications of these studies remain unclear. Unlike H₂O₂+Fe³⁺, XO+P does not seem to affect sarcolemmal Ca²⁺ channels or SR Ca²⁺ release if its presence is transient, as in this study. Nevertheless, such brief exposures did suffice to reduce force development by decreasing the Ca²⁺ sensitivity of the myofilaments. The effect of XO+P treatment on the myofilaments could be direct, as demonstrated in skinned cardiac fibers,^{11 12} an effect that may reflect inhibition of myofibrillar ATPase activity.³⁰ It is not clear from these previous studies whether such direct effects would persist after the removal of XO+P for 30 minutes. The increase in diastolic [Ca²⁺]_i produced by XO+P (Fig 7) itself may suffice to produce stunning.³⁹ Another possibility is that the XO+P system induces the production of oxidized glutathione via generation of ·O₂^-, recruiting a defense mechanism that the cell uses to minimize oxidative stress.⁴⁰ Bauer et al⁴¹ showed that oxidized glutathione (4 mmol/L) increases Ca₅₀ (0.25 pCa units) without affecting F_max. In addition, the amount of reduced glutathione, an endogenous Ca²⁺ sensitizer,⁴¹ may be decreased at the same time. Thus, the synergistic effect of the decrease of reduced glutathione and increase of oxidized glutathione could contribute to the observed decrease in myofilament Ca²⁺ sensitivity.

Relative Roles of ·OH and ·O₂^- in Myocardial Stunning
There is much evidence that OFRs play an important role in myocardial stunning,¹ but the precise cellular mechanism by which OFRs foster stunning is not yet clear. The above-mentioned studies do not address this question directly, especially given the fact that most OFRs are produced only transiently (for a few minutes) at the beginning of reperfusion,^{2 3 4} whereas most of those studies were obtained in the continuous presence of ·OH and/or ·O₂^-. Direct measurements of [Ca²⁺]_i with NMR⁴² or aequorin⁴³ have revealed an elevation of [Ca²⁺]_i in the early postischemic period, coincident with the period of the burst of OFR generation.^{2 3 4} Other studies have shown that transient exposure to H₂O₂+Fe³⁺ results in increased [Ca²⁺]_i, although only in association with much more severe damage than that produced in the present study.^{17 44} Thus, it appears that OFRs may mediate myocardial stunning at least partially by causing Ca²⁺ overload. We found that brief treatment with the XO+P-generating system could mimic some important features of stunned myocardium: no deficit of activator Ca²⁺, but decreased Ca²⁺ sensitivity. Furthermore, diastolic [Ca²⁺]_i increased during the treatment with XO+P. In the XO+P system, the primary free radical generated is ·O₂^-, but we cannot rule out the possibility that ·OH was also produced, since we did not measure OFR production in our study. Thus, it is possible that the differential effects of the two OFR-generating systems could have been due to the differences in the quantities of ·OH produced. This possibility seems unlikely, given that the differences in the effects are qualitative rather than quantitative. In addition, we have tested different concentrations of H₂O₂ in the H₂O₂+Fe³⁺ system and found either no effect of the treatment at lower concentrations (or shorter exposures) or no recovery at all at high concentrations or after longer exposures (results not shown). At no concentrations did we observe effects similar to those of XO+P treatment. Thus, the distinctive consequences of XO+P appear to reflect unique effects of ·O₂^-.

The XO+P system, which produces primarily ·O₂^-, mimics faithfully many of the phenotypic features of stunned myocardium. Nevertheless, the distinct effect of transient exposure to H₂O₂+Fe³⁺ as opposed to XO+P does not exclude a role of ·OH in causing myocardial stunning. Hydroxyl radical has recently been shown to be the predominant OFR species during reperfusion,^{1 10} and numerous studies have shown that ·OH scavengers and the iron chelator desferoxamine attenuate stunning.^{9 45 46} Our observation that exposure to an ·OH-generating system does not faithfully mimic stunning suggests that ·OH is not the only OFR involved in stunning. In fact, superoxide dismutase alone has considerable benefit,⁴⁷ supporting an important role for ·O₂^-. Combined antioxidant interventions that include superoxide dismutase, an ·OH scavenger, catalase, and desferoxamine offered the best protection against stunning.¹⁰ Both H₂O₂+Fe³⁺ and XO+P modestly reduced maximal Ca²⁺-activated force. Although individually insignificant, the combined depressant effects of the two types of radicals (·OH and ·O₂^-) on F_max may become quite significant during genuine ischemia and reperfusion; indeed, such an additive effect may underlie the depression of F_max in stunned myocardium.¹⁴

	Selected Abbreviations and Acronyms

 

[Ca2+]i = intracellular [Ca2+] with fura-2 H2O2+Fe3+ = H2O2+Fe3+-nitrilotriacetic acid KH = Krebs-Henseleit NTA = nitrilotriacetic acid OFR = oxygen free radical SR = sarcoplasmic reticulum XO+P = xanthine oxidase+purine

	Acknowledgments

 
This study was supported by the NIH (R01-HL-44065).

Received March 27, 1996; revision received June 8, 1996; accepted June 16, 1996.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Bolli R. Mechanism of myocardial `stunning.' Circulation. 1990;82:723-738.[Abstract/Free Full Text]
   
2. Garlick PB, Davies MJ, Hearse DJ, Slater TF. Direct detection of free radicals in the reperfused rat heart using electron spin resonance spectroscopy. Circ Res. 1987;61:757-760.[Abstract/Free Full Text]
   
3. Bolli R, Patel BS, Jeroudi MO, Lai EK, McCay PB. Demonstration of free radical generation in `stunned' myocardium of intact dogs with the use of the spin trap alpha-phenyl N-tert-butyl nitrone. J Clin Invest. 1988;82:476-485. [Published erratum appears in J Clin Invest. 1988;82:following 1807.]
   
4. Zweier JL, Kuppusamy P, Williams R, Rayburn BK, Smith D, Weisfeldt ML, Flaherty JT. Measurement and characterization of postischemic free radical generation in the isolated perfused heart. J Biol Chem. 1989;264:18890-18895.[Abstract/Free Full Text]
   
5. Myers ML, Bolli R, Lekich RF, Hartley CJ, Roberts R. N-2-Mercaptopropionylglycine improves recovery of myocardial function after reversible regional ischemia. J Am Coll Cardiol. 1986;8:1161-1168.[Abstract]
   
6. Przyklenk K, Kloner RA. Superoxide dismutase plus catalase improve contractile function in the canine model of the `stunned myocardium.' Circ Res. 1986;58:148-156.[Abstract/Free Full Text]
   
7. Bolli R, Zhu WX, Hartley CJ, Michael LH, Repine JE, Hess ML, Kukreja RC, Roberts R. Attenuation of dysfunction in the postischemic `stunned' myocardium by dimethylthiourea. Circulation. 1987;76:458-468.[Abstract/Free Full Text]
   
8. Bolli R, Jeroudi MO, Patel BS, Aruoma OI, Halliwell B, Lai EK, McCay PB. Marked reduction of free radical generation and contractile dysfunction by antioxidant therapy begun at the time of reperfusion: evidence that myocardial `stunning' is a manifestation of reperfusion injury. Circ Res. 1989;65:607-622.[Abstract/Free Full Text]
   
9. Sun JZ, Kaur H, Halliwell B, Li XY, Bolli R. Use of aromatic hydroxylation of phenylalanine to measure production of hydroxyl radicals after myocardial ischemia in vivo: direct evidence for a pathogenetic role of the hydroxyl radical in myocardial stunning. Circ Res. 1993;73:534-549.[Abstract/Free Full Text]
   
10. Mekfi M, Veksler V, Mateo P, Maupoil V, Rochette L, Ventura-Clapier R. Creatine kinase is the main target of reactive oxygen species in cardiac myofibrils. Circ Res. 1996;78:1016-1027.[Abstract/Free Full Text]
    
11. MacFarlane NG, Miller DJ. Depression of peak force without altering calcium sensitivity by the superoxide anion in chemically skinned cardiac muscle of rat. Circ Res. 1992;70:1217-1224.[Abstract/Free Full Text]
    
12. Lowe H, Baeger I, Blasig IE, Haseloff RF. Oxygen radicals attenuate the contractility of skinned muscle fibres from the pig myocardium. Pharmazie. 1994;49:845-849.[Medline] [Order article via Infotrieve]
    
13. Kusuoka H, Porterfield JK, Weisman HF, Weisfeldt ML, Marban E. Pathophysiology and pathogenesis of stunned myocardium: depressed Ca²⁺ activation of contraction as a consequence of reperfusion-induced cellular calcium overload in ferret hearts. J Clin Invest. 1987;79:950-961.
    
14. Gao WD, Atar D, Backx PH, Marban E. Relationship between intracellular calcium and contractile force in stunned myocardium: direct evidence for decreased myofilament Ca²⁺ responsiveness and altered diastolic function in intact ventricular muscle. Circ Res. 1995;76:1036-1048.[Abstract/Free Full Text]
    
15. Backx PH, ter Keurs HE. Fluorescent properties of rat cardiac trabeculae microinjected with fura-2 salt. Am J Physiol. 1993;264(pt 2):H1098-H1110.
    
16. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca²⁺ indicators with greatly improved fluorescent properties. J Biol Chem. 1985;260:3440-3450.[Abstract/Free Full Text]
    
17. Corretti MC, Koretsune Y, Kusuoka H, Chacko VP, Zweier JL, Marban E. Glycolytic inhibition and calcium overload as consequences of exogenously generated free radicals in rabbit hearts. J Clin Invest. 1991;88:1014-1025.
    
18. Burton KP, McCord JM, Ghai G. Myocardial alterations due to free-radical generation. Am J Physiol. 1984;246(pt 2):H776-H783.
    
19. Blaustein AS, Schine L, Brooks WW, Fanburg BL, Bing OH. Influence of exogenously generated oxidant species on myocardial function. Am J Physiol. 1986;250(pt 2):H595-H599.
    
20. Snedecor GW, Cochran WG. Statistical Methods. Ames, Iowa: Iowa State University Press; 1989.
    
21. Winer BJ. Statistical Principles in Experimental Design. New York, NY: McGraw-Hill Inc; 1982.
    
22. ter Keurs HE, Bucx JJJ, Harmsen E, de Tombe PP, Leijendekker WJ. Ischemia, high-energy phosphate metabolism and sarcomere dynamics in myocardium. In: de Jong JW, ed. Myocardial Energy Metabolism. Dordrecht, Netherlands: Martinus Nijhoff Publishers; 1988:181-194.
    
23. Kusuoka H, Marban E. Cellular mechanisms of myocardial stunning. Annu Rev Physiol. 1992;54:243-256.[Medline] [Order article via Infotrieve]
    
24. Kitakaze M, Weisman HF, Marban E. Contractile dysfunction and ATP depletion after transient calcium overload in perfused ferret hearts. Circulation. 1988;77:685-695.[Abstract/Free Full Text]
    
25. Koretsune Y, Marban E. Cell calcium in the pathophysiology of ventricular fibrillation and in the pathogenesis of post-arrhythmic contractile dysfunction. Circulation. 1989;80:369-379.[Abstract/Free Full Text]
    
26. Kort AA, Lakatta EG, Marban E, Stern MD, Wier WG. Fluctuations in intracellular [Ca²⁺] and their effect on tonic tension in canine cardiac Purkinje fibres. J Physiol. 1985;367:291-308.[Abstract/Free Full Text]
    
27. Okabe E, Hess ML, Oyama M, Ito H. Characterization of free radical-mediated damage of canine cardiac sarcoplasmic reticulum. Arch Biochem Biophys. 1983;225:164-177.[Medline] [Order article via Infotrieve]
    
28. Kaneko M, Beamish RE, Dhalla NS. Depression of heart sarcolemmal Ca²⁺-pump activity by oxygen free radicals. Am J Physiol. 1989;256(pt 2):H368-H374.
    
29. Kim MS, Akera T. O₂ free radicals: cause of ischemia-reperfusion injury to cardiac Na⁺-K⁺-ATPase. Am J Physiol. 1987;252(pt 2):H252-H257.
    
30. Ventura C, Guarnieri C, Caldarera CM. Inhibitory effect of superoxide radicals on cardiac myofibrillar ATPase activity. Ital J Biochem. 1985;34:267-274.[Medline] [Order article via Infotrieve]
    
31. Goldhaber JI, Ji S, Lamp ST, Weiss JN. Effects of exogenous free radicals on electromechanical function and metabolism in isolated rabbit and guinea pig ventricle: implications for ischemia and reperfusion injury. J Clin Invest. 1989;83:1800-1809.
    
32. Coetzee WA, Opie LH. Effects of oxygen free radicals on isolated cardiac myocytes from guinea-pig ventricle: electrophysiological studies. J Mol Cell Cardiol. 1992;24:651-663.[Medline] [Order article via Infotrieve]
    
33. Shi ZQ, Davison AJ, Tibbits GF. Effects of active oxygen generated by DTT/Fe²⁺ on cardiac Na⁺/Ca²⁺ exchange and membrane permeability to Ca²⁺. J Mol Cell Cardiol. 1989;21:1009-1016.[Medline] [Order article via Infotrieve]
    
34. Baas AS, Berk BC. Differential activation of mitogen-activated protein kinases by H₂O₂ and O₂^- in vascular smooth muscle cells. Circ Res. 1995;77:29-36.[Abstract/Free Full Text]
    
35. Khalid MA, Ashraf M. Direct detection of endogenous hydroxyl radical production in cultured adult cardiomyocytes during anoxia and reoxygenation: is the hydroxyl radical really the most damaging radical species? Circ Res. 1993;72:725-736.[Abstract/Free Full Text]
    
36. Fabiato A. Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am J Physiol. 1983;245:C1-C14.[Abstract/Free Full Text]
    
37. Holmberg SR, Cumming DV, Kusama Y, Hearse DJ, Poole-Wilson PA, Shattock MJ, Williams AJ. Reactive oxygen species modify the structure and function of the cardiac sarcoplasmic reticulum calcium-release channel. Cardioscience. 1991;2:19-25.[Medline] [Order article via Infotrieve]
    
38. MacFarlane NG, Miller DJ, Smith GL, Steele DS. Effects of oxidants on the sarcoplasmic reticulum of saponin treated rat ventricular trabeculae. Cardiovasc Res. 1994;28:1647-1652.[Medline] [Order article via Infotrieve]
    
39. Marban E. Myocardial stunning and hibernation: the physiology behind the colloquialisms. Circulation. 1991;83:681-688.[Free Full Text]
    
40. Ferrari R, Ceconi C, Curello S, Cargnoni A, Alfieri O, Pardini A, Marzollo P, Visioli O. Oxygen free radicals and myocardial damage: protective role of thiol-containing agents. Am J Med. 1991;91:95S-105S.[Medline] [Order article via Infotrieve]
    
41. Bauer SF, Schwarz K, Ruegg JC. Glutathione alters calcium responsiveness of cardiac skinned fibers. Basic Res Cardiol. 1989;84:591-596.[Medline] [Order article via Infotrieve]
    
42. Marban E, Kitakaze M, Koretsune Y, Yue DT, Chacko VP, Pike MM. Quantification of [Ca²⁺]_i in perfused hearts: critical evaluation of the 5F-BAPTA and nuclear magnetic resonance method as applied to the study of ischemia and reperfusion. Circ Res. 1990;66:1255-1267.[Abstract/Free Full Text]
    
43. Carrozza JP Jr, Bentivegna LA, Williams CP, Kuntz RE, Grossman W, Morgan JP. Decreased myofilament responsiveness in myocardial stunning follows transient calcium overload during ischemia and reperfusion. Circ Res. 1992;71:1334-1340.[Abstract/Free Full Text]
    
44. Josephson RA, Silverman HS, Lakatta EG, Stern MD, Zweier JL. Study of the mechanisms of hydrogen peroxide and hydroxyl free radical-induced cellular injury and calcium overload in cardiac myocytes. J Biol Chem. 1991;266:2354-2361.[Abstract/Free Full Text]
    
45. Farber NE, Vercellotti GM, Jacob HS, Pieper GM, Gross GJ. Evidence for a role of iron-catalyzed oxidants in functional and metabolic stunning in the canine heart. Circ Res. 1988;63:351-360.[Abstract/Free Full Text]
    
46. Sekili S, McCay PB, Li XY, Zughaib M, Sun JZ, Tang L, Thornby JI, Bolli R. Direct evidence that the hydroxyl radical plays a pathogenetic role in myocardial `stunning' in the conscious dog and demonstration that stunning can be markedly attenuated without subsequent adverse effects. Circ Res. 1993;73:705-723.[Abstract/Free Full Text]
    
47. Schrier GM, Hess ML. Quantitative identification of superoxide anion as a negative inotropic species. Am J Physiol. 1988;255(pt 2):H138-H143.
    

This article has been cited by other articles:

				I. A. Williams and D. G. Allen The role of reactive oxygen species in the hearts of dystrophin-deficient mdx mice Am J Physiol Heart Circ Physiol, September 1, 2007; 293(3): H1969 - H1977. [Abstract] [Full Text] [PDF]

				M. Seddon, Y. H Looi, and A. M Shah Oxidative stress and redox signalling in cardiac hypertrophy and heart failure Heart, August 1, 2007; 93(8): 903 - 907. [Abstract] [Full Text] [PDF]

				D. K. Glover, M. Ruiz, K. Takehana, F. D. Petruzella, J. M. Rieger, T. L. Macdonald, D. D. Watson, J. Linden, and G. A. Beller Cardioprotection by adenosine A2A agonists in a canine model of myocardial stunning produced by multiple episodes of transient ischemia Am J Physiol Heart Circ Physiol, June 1, 2007; 292(6): H3164 - H3171. [Abstract] [Full Text] [PDF]

				R. Liu, J. L. Garvin, Y. Ren, P. J. Pagano, and O. A. Carretero Depolarization of the macula densa induces superoxide production via NAD(P)H oxidase Am J Physiol Renal Physiol, June 1, 2007; 292(6): F1867 - F1872. [Abstract] [Full Text] [PDF]

				T. Dai, Y. Tian, C. G. Tocchetti, T. Katori, A. M. Murphy, D. A. Kass, N. Paolocci, and W. D. Gao Nitroxyl increases force development in rat cardiac muscle J. Physiol., May 1, 2007; 580(3): 951 - 960. [Abstract] [Full Text] [PDF]

				C. Doerries, K. Grote, D. Hilfiker-Kleiner, M. Luchtefeld, A. Schaefer, S. M. Holland, S. Sorrentino, C. Manes, B. Schieffer, H. Drexler, et al. Critical Role of the NAD(P)H Oxidase Subunit p47phox for Left Ventricular Remodeling/Dysfunction and Survival After Myocardial Infarction Circ. Res., March 30, 2007; 100(6): 894 - 903. [Abstract] [Full Text] [PDF]

				N. Hiranandani, T. Bupha-Intr, and P. M. L. Janssen SERCA overexpression reduces hydroxyl radical injury in murine myocardium Am J Physiol Heart Circ Physiol, December 1, 2006; 291(6): H3130 - H3135. [Abstract] [Full Text] [PDF]

				C. E. Murdoch, M. Zhang, A. C. Cave, and A. M. Shah NADPH oxidase-dependent redox signalling in cardiac hypertrophy, remodelling and failure Cardiovasc Res, July 15, 2006; 71(2): 208 - 215. [Abstract] [Full Text] [PDF]

				D. J. Grieve, J. A. Byrne, A. Siva, J. Layland, S. Johar, A. C. Cave, and A. M. Shah Involvement of the Nicotinamide Adenosine Dinucleotide Phosphate Oxidase Isoform Nox2 in Cardiac Contractile Dysfunction Occurring in Response to Pressure Overload J. Am. Coll. Cardiol., February 21, 2006; 47(4): 817 - 826. [Abstract] [Full Text] [PDF]

				L. B. Stull, M. K. Leppo, L. Szweda, W. D. Gao, and E. Marban Chronic Treatment With Allopurinol Boosts Survival and Cardiac Contractility in Murine Postischemic Cardiomyopathy Circ. Res., November 12, 2004; 95(10): 1005 - 1011. [Abstract] [Full Text] [PDF]