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(Circulation. 1999;100:346-353.)
© 1999 American Heart Association, Inc.

Clinical Investigation and Reports

Effect of ß-Blockers on Free Radical–Induced Cardiac Contractile Dysfunction

M. Flesch, MD; C. Maack, MS; B. Cremers, MS; A. T. Bäumer, MD; M. Südkamp, MD; M. Böhm, MD

From the Klinik III für Innere Medizin and Klinik für Herz- und Thoraxchirurgie (M.S.) der Universität zu Köln, Cologne, Germany. Dr Flesch and C. Maack contributed equally to the article.

Correspondence to M. Flesch, MD, Joseph-Stelzmann-Straße 9, 50924 Cologne, Germany.

	Abstract

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Background—We examined the effects of hydroxyl radicals (OH·) on human myocardial contractility and on sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) activity and the effects of the ß-receptor antagonists metoprolol, carvedilol, and its metabolite BM-910228.

Methods and Results—Isometric force of contraction was determined in isolated human myocardium. H₂O₂ 1 mmol/L and Fe³⁺-nitrilotriacetic acid (Fe³⁺-NTA) 0.1 mmol/L used for generation of OH· induced a decrease in basal force of contraction and an increase in diastolic tension in atrial and left ventricular myocardial preparations. After challenge with OH·, the maximum positive inotropic response to Ca²⁺ 1.8 to 15 mmol/L was decreased by 60% and by 39%, respectively. The effects of OH· could be blocked by catalase. Carvedilol and its metabolite BM-910228 attenuated the OH·-induced impairment of the inotropic response to Ca²⁺ in atrial myocardial preparations. Metoprolol had no significant effect. The stimulation frequency (0.5 to 3.0 Hz)–dependent increase in force of contraction and decrease in diastolic tension were abolished after exposure of atrial trabeculae to OH·. In parallel, SERCA activity was decreased by OH· concentration-dependently, as determined in myocardial membrane preparations. BM-910228 partially restored the force-frequency relationship and preserved SERCA activity.

Conclusions—OH· radicals induce an impairment of contraction and relaxation and an attenuation of the force-frequency relationship in human myocardium accompanied by an inhibition of SERCA. Carvedilol and BM-910228 partly prevented OH·-induced contractile dysfunction. These observations could explain the improvement of ejection fraction in heart failure trials with carvedilol without a restoration of ß-adrenergic receptor density.

Key Words: heart failure • contractility • free radicals • receptors, adrenergic, beta

	Introduction

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In chronic heart failure,¹ doxorubicin-induced cardiomyopathy,² and myocardial ischemia-reperfusion injury,^{3 4} oxygen-derived free radicals might contribute to cardiac contractile dysfunction. In vitro studies on rat cardiac myocytes⁵ and papillary muscles⁶ have demonstrated that oxygen-derived free radicals lead to a decrease in systolic cell shortening and force of contraction and to an increase in diastolic tension. The mechanisms by which free radicals impair cardiac contractility are not completely elucidated. In particular, few data are derived from human myocardium.

In patients with chronic heart failure, treatment with ß-adrenergic receptor antagonists leads to a reduction in mortality and an improvement of left ventricular function.^{7 8 9} The mechanisms by which ß-adrenergic receptor blockers exert their beneficial effects are unclear. One possible mechanism is the restoration of the ß-adrenergic receptor–adenylate cyclase signal transduction pathway in the failing heart, in which, because of chronic excessive adrenergic innervation, ß₁-adrenergic receptors are downregulated¹⁰ and inhibitory guanine nucleotide binding proteins are upregulated.¹¹ In this context, Gilbert et al¹² demonstrated similar effects of metoprolol and carvedilol on ejection fraction. However, only metoprolol, but not carvedilol, led to an increase in ß-adrenergic receptor density. Thus, there might be other mechanisms that contribute to the beneficial effects of carvedilol.

Carvedilol is a vasodilating ß-blocker with potent antioxidant activity.¹³ It has been demonstrated to reduce infarct size¹⁴ and to prevent myocardial ischemia-reperfusion–induced apoptosis¹⁵ in animal models of myocardial infarction. Because ischemia-reperfusion–induced apoptosis is related to enhanced release of oxygen-derived free radicals,¹⁶ the latter effect of carvedilol might depend on its antioxidative properties.¹⁵ These properties of carvedilol might also have been of relevance in recent heart failure trials.^{9 12} Because increased release of free radicals in hearts from patients with chronic heart failure¹⁷ might also contribute to apoptosis in failing human myocardium,^{18 19} one might speculate whether carvedilol could prevent apoptosis in this pathological condition and, in particular, whether carvedilol increased cardiac ejection fraction in heart failure trials,^{9 12} because it inhibited the effects of oxygen-derived free radicals on cardiac contractility.¹⁷

The present study concentrated on the latter hypothesis. The effects of hydroxyl (OH·) free radicals on human myocardial contractility were examined, and the question was addressed whether these effects can be prevented by carvedilol and the carvedilol metabolite BM-910228. Also, the effects of metoprolol were studied. OH· free radicals were chosen for the experiments because they have been suggested to be the predominant oxidant species causing cellular injury.^{20 21 22} The preventive effects of the carvedilol metabolite BM-910228 were examined because it is characterized by a lower ß-adrenergic receptor affinity but a 3-fold higher antioxidative capacity than carvedilol.²³ Thus, the use of the carvedilol metabolite BM-910228 allowed us to differentiate between the effects of carvedilol that are due to its antioxidative activity and those that are due to its ß-receptor blocking properties.

	Methods

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Human Myocardial Tissue
Experiments were performed on human right atrial trabeculae obtained during open-heart bypass surgery and on left ventricular papillary muscle preparations from nonfailing human hearts obtained during cardiac transplantation. Bypass patients (mean age, 65±1 years; 46 male, 21 female) had no signs of atrial dilatation or arrhythmias or of left ventricular dysfunction. Nonfailing donor hearts were hearts that were used for mitral and aortic valve allograft preparation because there was no appropriate recipient available for cardiac transplantation. The mean age of organ donors (3 male, 2 female) was 35±8 years. There were no signs of left ventricular dysfunction in these patients.

ß-Adrenergic Receptor Binding Studies
ß-Adrenergic receptors in left ventricular myocardial membrane preparations were investigated by use of [¹²⁵I]iodocyanopindolol ([¹²⁵I]ICYP) as described previously.²⁴ The IC₅₀ values were determined from computer-fitted regression analysis (Graph Pad Prism), and K_D values were calculated according to the method of Cheng and Prussoff.²⁵

Determination of Force of Contraction in Isolated Human Myocardium
Isometric force of contraction was determined on isolated electrically driven muscle preparations as described previously.²⁶ Briefly, atrial trabeculae (1 to 2 mm wide, 4 to 6 mm long) and left ventricular papillary muscle strips (1 to 2 mm wide, 7 to 9 mm long) were electrically stimulated (frequency, 1 Hz; impulse duration, 5 ms; intensity, 10% to 20% greater than threshold) in a modified Tyrode's solution maintained at 37°C and aerated with 95% O₂ and 5% CO₂. Muscles were stretched to the length at which force of contraction was maximal.

Determination of Ca²⁺-ATPase Activity in Atrial Sarcoplasmic Reticulum Membranes
For determination of sarcoplasmic reticulum (SR) Ca²⁺-ATPase (SERCA) activity, atrial SR membranes were prepared according to Meissner and Henderson²⁷ and Sitsapesan and Williams.²⁸ Myocardial tissue was chilled in ice-cold homogenization buffer (in mmol/L: sucrose 300, PMSF 1, and PIPES 20, pH 7.4). Connective tissue was trimmed away, and myocardial tissue was homogenized. The homogenate was spun at 1920g (Beckman J-218) for 20 minutes. The supernatant was filtered and centrifuged at 100 000g for 60 minutes (Beckman JA 20). The pellet was resuspended in a 10% sucrose buffer containing (in mmol/L) KCl 400, MgCl₂ 0.5, CaCl₂ 0.5, EGTA 0.5, and PIPES 25, pH 7.0. SERCA activity, defined as hydrolysis of Mg²⁺-ATP in ADP plus inorganic phosphorus (P_i) in the presence of Ca²⁺, was determined according to Kyte²⁹ and Xu et al.³⁰ SR preparations (final concentration, 50 µg/mL) were suspended in (in mmol/L) MOPS 21, NaN₃ 4.9, EGTA 0.06, KCl 100, and MgCl₂ 3, and Ca²⁺-ionophore A23187 1 µmol/L. CaCl₂ was added to the reaction to yield the desired free Ca²⁺ concentrations calculated according to Fabiato.³¹ The reaction was carried out in 1 mL at 30°C for 25 minutes after having been started with ATP 1 mmol/L. Reaction mixture (50 µL) was added to 500 µL of a phosphorus reaction mixture (Sigma Chemicals) consisting of 0.4 mmol/L of ammonium molybdate in sulfuric acid. Production of P_i was measured by spectrophotometric (340 nm) determination of unreduced phosphomolybdate complex. Basal activity was measured in the absence of Ca²⁺ and in the presence of EGTA 4 mmol/L simultaneously. Experiments were carried out in triplicate.

Hydroxyl Radical–Generating System and Experimental Protocol
OH· radicals were generated via the Haber-Weiss-Fenton reaction from H₂O₂ 1 mmol/L in the presence of Fe³⁺-nitrilotriacetic acid (Fe³⁺-NTA) 0.1 mmol/L.³² These concentrations generate a magnitude of OH· similar to that observed during the early minutes of postischemic reperfusion.^{30 32 33} For investigation of the effects of OH· on basal and Ca²⁺-stimulated force of contraction, myocardial preparations were exposed to H₂O₂ 1 mmol/L and Fe³⁺ 100 µmol/L for 15 minutes. After the bathing solution was exchanged, preparations were allowed to equilibrate for 20 minutes before the inotropic response to extracellular Ca²⁺ 1.8 to 15 mmol/L was determined. This approach was chosen because it mimics physiological situations in which contractile dysfunction persists longer than free radical exposure, which may last only for a few minutes.³⁴ The effects of OH· on the force-frequency relationship (0.5 to 3 Hz) were examined in atrial trabeculae by determination of 3 consecutive force-frequency relationships: before the exposure of the myocardium to OH·, in the presence of OH·, and after washout of free radicals. Before the onset of the experiment, myocardial preparations were preincubated with either catalase 120 U/min, carvedilol 1 nmol/L, BM-910228 1 µmol/L, metoprolol 30 nmol/L, or vehicle for 50 minutes (basal contractility, inotropic response to Ca²⁺) or for 30 minutes (force-frequency relationship).

Materials
Chemicals were from Sigma Chemical Co. Radioactive ligands were from Amersham.

Statistics
Data shown are mean±SEM. Statistical significance was analyzed by ANOVA (least significant difference). When ANOVA could not be applied, the Wilcoxon or Mann-Whitney test was used. A value of P<0.05 was considered significant.

	Results

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ß-Adrenergic Receptor Binding and Antiadrenergic Effects of Carvedilol and BM-910228
To examine whether carvedilol and its metabolite BM-910228 differ with respect to their ß-adrenergic receptor affinities, competitive binding experiments of carvedilol and BM-910228 with [¹²⁵I]ICYP were performed in isolated human ventricular membranes. ß-Adrenergic receptor affinity of BM-910228 was 400-fold lower than with carvedilol (K_i values: 326 [253 to 421] nmol/L versus 0.82 [0.61 to 1.11] nmol/L, P<0.01) (Figure 1). Both carvedilol and BM-910228 decreased isoprenaline-enhanced force of contraction concentration-dependently, but the antiadrenergic potency of BM-910228 was 100-fold less pronounced than the potency of carvedilol (not shown). For the following experiments, BM-910228 and carvedilol were used at concentrations at which ß-adrenergic receptor occupations by the antagonists were comparable (1 µmol/L and 1 nmol/L, respectively).

View larger version (21K): [in this window] [in a new window]   Figure 1. Competition of carvedilol (n=5) and its metabolite BM-910228 (n=3) with binding of 125I-labeled ICYP to human left ventricular myocardial membranes.

Effect of OH· on Basal Force of Contraction
The effect of Fe³⁺-NTA 0.1 mmol/L and H₂O₂ 1 mmol/L on myocardial contractility is demonstrated in Figure 2. There was a decrease in systolic force of contraction over a period of 10 minutes, followed by a rise in diastolic tension. After a change to radical-free Tyrode's solution, diastolic tension returned to basal values, whereas systolic force of contraction remained reduced. Effects of OH· free radicals were similar in atrial trabeculae and in left ventricular myocardial preparations (Figure 3 and Table 1). Catalase prevented the effect of OH· on force of contraction. Preincubation of atrial trabeculae with metoprolol led to a more pronounced decrease in force of contraction than carvedilol or its metabolite BM-910228 over 50 minutes of preincubation time (Table 1). The presence of ß-blockers had no significant effects on the acute decrease of force of contraction (Table 1) and on the rise of diastolic tension in response to OH· (data not shown).

View larger version (85K): [in this window] [in a new window]   Figure 2. Original tracing demonstrating effect of H2O2 and Fe3+-NTA on force of contraction in isolated human atrial trabeculae. Top lane shows control experiment in presence of catalase; bottom lane, effect in absence of catalase.

View larger version (24K): [in this window] [in a new window]   Figure 3. Mean values (±SEM) for effect of H2O2 and Fe3+-NTA on basal force of contraction (A) and diastolic tension (B) in human right atrial and left ventricular trabeculae. C, Inotropic response to external Ca2+ after washout of OH· free radicals.

View this table: [in this window] [in a new window]   Table 1. Effect of H2O2 and Fe3+-NTA on Basal Force of Contraction (FOC) and the Maximum Inotropic Response to Ca2+ 15 mmol/L in Human Right Atrial and Left Ventricular Trabeculae

Effect of OH· Free Radicals on the Inotropic Response to Ca²⁺
The concentration-dependent inotropic response to extracellular Ca²⁺ was determined after washout of OH· radicals. In atrial and ventricular control preparations, Ca²⁺ caused a concentration-dependent increase in force of contraction by maximally 334% and 322% of basal values, respectively (Figure 3). After free radical exposure, the effect was significantly attenuated. The effects of OH· radicals could be prevented by preincubation of atrial trabeculae with catalase but similarly also by preincubation with carvedilol and BM-910228 (Table 1, Figure 4). Metoprolol had no significant effect.

View larger version (25K): [in this window] [in a new window]   Figure 4. Maximum inotropic effect of CaCl2 15 mmol/L on control atrial myocardial preparations (control), atrial myocardium exposed for 15 minutes to Fe3+-NTA/H2O2, and myocardium exposed to Fe3+-NTA/H2O2 after preincubation with metoprolol (Meto), carvedilol (Carv), or BM-910228 (BM) and in presence of catalase (Cat).

Effect of OH· Free Radicals and ß-Blockers on the Force-Frequency Relationship
The effects of OH· radicals on the force-frequency relationship were determined in atrial trabeculae. An increase in stimulation frequency (0.5 to 3 Hz) induced a significant increase in force of contraction and a frequency-dependent decrease in diastolic tension (Figures 5A and 6A, Table 2). Exposure to OH· radicals abolished this frequency-dependent increase in force of contraction (Figure 5B) and led to a frequency-dependent increase in diastolic tension (Figure 6B). The impairment of the force-frequency relationship lasted even after washout of OH· (Figures 5C and 6C). In contrast, in trabeculae preincubated with BM-910228, the force-frequency relationship significantly recovered after washout of OH· free radicals. In carvedilol-pretreated myocardium, there was a trend toward a partial restoration of the force-frequency relationship (Table 2). In contrast, metoprolol preincubation did not lead to a recovery of the force-frequency relationship (Figures 5C and 6C, Table 2). Parallel to the frequency-dependent increase in force of contraction, peak rate of tension rise and decay increased. In the presence of and after removal of Fe³⁺/H₂O₂, these parameters were significantly attenuated. Preincubation with BM-910228 led to an improvement of both parameters (Table 2).

View larger version (22K): [in this window] [in a new window]   Figure 5. Effect of OH· free radicals on force-frequency relationship in human atrial trabeculae. Experiments were performed either under control conditions or after preincubation with catalase, BM-910228, or metoprolol. A, Force-frequency relationship before addition of Fe3+-NTA/H2O2; B, in presence and C, after washout of Fe3+-NTA/H2O2.

View larger version (21K): [in this window] [in a new window]   Figure 6. Effect of OH· free radicals on frequency-dependent development of diastolic tension in human atrial trabeculae. Experiments were performed either under control conditions or after preincubation with catalase, BM-910228, or metoprolol. A, Frequency-dependent alterations in diastolic tension before addition of Fe3+-NTA/H2O2; B, in presence and C, after washout of Fe3+-NTA/H2O2.

View this table: [in this window] [in a new window]   Table 2. Effect of H2O2 and Fe3+-NTA on Frequency-Dependent Changes in Contraction Parameters in Human Right Atrial Trabeculae

Effect of OH· on SERCA Activity
SERCA activity in isolated SR membranes was dependent on the free Ca²⁺ concentration in the reaction mixture (0.04 to 35 µmol/L). Maximum activity was 124.2±10.5 nmol ATP/mg proteinxmin at a free Ca²⁺ concentration of 1.09 µmol/L (EC₅₀ value, 0.1 [0.03 to 0.38] µmol/L). Experiments were carried out at this maximum effective Ca²⁺ concentration. Figure 7A illustrates the concentration-dependent decrease of SERCA activity by increasing concentrations of Fe³⁺-NTA (20 to 200 µmol/L) in the presence of 1 mmol/L of H₂O₂ to maximally 53% of control values (n=4, P<0.05). All further experiments were performed at submaximal concentrations of OH· free radicals (H₂O₂, 1 mmol/L; Fe³⁺-NTA, 60 µmol/L), at which SERCA activity was inhibited by 31±4% (n=5, P<0.05 versus control). At this concentration, the effects of OH· radicals on SERCA activity were significantly attenuated in the presence of carvedilol 0.1 µmol/L and BM-910228 0.001 µmol/L (Figure 7B).

View larger version (21K): [in this window] [in a new window]   Figure 7. A, Concentration-dependent effect of H2O2 1 mmol/L and Fe3+-NTA 20 to 200 µmol/L on Ca2+-ATPase activity in isolated SR membrane preparations from human right atrial myocardium (n=5). B, Maximum SERCA activity under control conditions, in presence of H2O2 1 mmol/L alone, in presence of Fe3+-NTA/H2O2 (n=5), in presence of Fe3+-NTA/H2O2 after preincubation of SR membranes with carvedilol (Carv, n=5), and with BM-910228 (n=5), respectively.

	Discussion

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The present study demonstrated that exposure to OH· free radicals leads to a decrease in systolic force of contraction and an increase in diastolic tension in isolated human atrial and ventricular myocardium as well as a reduction of the inotropic responsiveness to extracellular Ca²⁺. Also, OH· free radicals induced an impairment of the positive force-frequency relationship.

Despite other possible mechanisms such as ATP deficiency or decreased Ca²⁺ sensitivity of the myofilaments,³ alterations in intracellular Ca²⁺ handling might be one major cause for cardiac contractile dysfunction induced by OH· radicals. Increased intracellular Ca²⁺ concentrations as a possible cause for an increase in end-diastolic pressure, diastolic tension, or myocyte contracture have been observed in Langendorff-perfused rabbit hearts after OH· radical treatment,³² in stunned rat ventricular trabeculae,⁶ and in isolated cardiac myocytes.⁵ Because of the similarity between the functional effects of OH· free radicals in rabbit and rat myocardium^{5 6 32} and effects observed in human myocardium in this study, one might assume that an increase in intracellular Ca²⁺ has also contributed to the development of contractile dysfunction and especially initial contracture in human tissue.

The importance of an altered intracellular Ca²⁺ homeostasis as one major cause of OH· free radical–induced contractile dysfunction in human myocardium is especially emphasized by the fact that OH· free radicals led to an impairment of the force-frequency relationship. In chronic heart failure, there is a strong correlation between an impaired force-frequency relationship and an impaired uptake of Ca²⁺ into the SR.^{35 36 37} Similarly, in this study, OH· free radicals not only abolished the frequency-dependent increase in systolic force of contraction but also led to a concentration-dependent decrease in SERCA activity. Thus, one might assume that OH· free radicals lead to decreased diastolic sequestration of Ca²⁺ because of SERCA inhibition, consequent to an impairment of relaxation and to an altered force-frequency relationship.

All effects of H₂O₂ plus Fe³⁺-NTA could be prevented by preincubation of myocardial preparations with catalase, which eliminated H₂O₂ by forming H₂O plus O₂. Also, carvedilol and BM-910228 restored the inotropic response to Ca²⁺ in OH· free radical–treated myocardium. This effect was more pronounced than the effect of the ß-blocker metoprolol, although because of sample sizes, the difference between carvedilol/BM-910228 and metoprolol did not reach statistical significance. Also, BM-910228 restored the positive force-frequency relationship in atrial myocardium after exposure to OH· radicals, and BM-910228 and carvedilol prevented the OH· radical–induced decrease in SERCA activity.

The mechanisms by which carvedilol and BM-910228 exert their protective effects remain speculative. Both pharmacological substances are potent OH· free radical scavengers¹³ that because of their lipophilicity might become enriched in myocardial membranes.²⁴ There, carvedilol and metabolites might exert antioxidant effects by interrupting continuously ongoing lipid peroxidation chains.²³ This hypothesized mechanism might explain why BM-910228–preincubated myocardium recovered better from OH· free radical–induced injury than nonpreincubated myocardium.

The demonstration of a preventive effect of carvedilol and BM-910228 on free radical–induced contractile dysfunction implies that the antioxidative activity of these substances could be of clinical relevance. Of course, one has to take into account that effects of ß- and -adrenergic receptor blockers on coronary and peripheral circulation or on heartbeat might also be important in the treatment of ischemic heart disease and chronic heart failure, which might be overlooked in the present experimental approach. Even more, chronic inotropism does not necessarily contribute to an increase in survival rate in patients with chronic heart failure.³⁸ However, like the suggestion that beneficial effects of carvedilol in the prevention of ischemia/reperfusion injury in vivo²³ and in vitro¹⁵ might be due to its antioxidative properties, one might assume that the beneficial effects of carvedilol in chronic heart failure⁹ are also partly mediated by prevention of free radical damage.

In summary, OH· free radicals induce severe contractile dysfunction in human myocardium, including development of contracture, decreased inotropic responsiveness to external Ca²⁺, and impairment of the force-frequency relationship. The beneficial effects of carvedilol and especially its metabolite BM-910228 on OH· free radical–induced contractile dysfunction in human myocardium underline the possible importance of the use of antioxidative substances in cardiac therapy and might help to explain different effects of specific ß-blockers used for treatment in chronic heart failure.

	Acknowledgments

 
This work was supported by the Deutsche Forschungsgemeinschaft (to Dr Böhm) and research grants from Byk-Gulden GmbH (Constance, Germany). Dr Flesch is a recipient of a Habilitations Scholarship of the Deutsche Forschungsgemeinschaft. The work contains parts of the doctoral thesis of C. Maack (University of Cologne).

Received December 31, 1998; revision received April 12, 1999; accepted April 28, 1999.

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