Novel Myofilament Ca²⁺-Sensitizing Property of Xanthine Oxidase Inhibitors

From the Section of Molecular and Cellular Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Md. The current address for Dr N.G. Pérez is Center for Cardiovascular Investigation, University of La Plata, La Plata, Argentina.

Correspondence to Eduardo Marbán, MD, PhD, Room 844, Ross Building, Johns Hopkins University School of Medicine, 720 Rutland Ave, Baltimore, MD 21205. E-mail marban{at}welchlink.welch.jhu.edu

	Abstract

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Abstract—Antioxidants are known to mitigate the cardiac contractile dysfunction that follows brief periods of ischemia ("myocardial stunning"). Stunning decreases contractility at the level of the contractile proteins; therefore, we asked whether antioxidant treatment preserves myofilament Ca²⁺ responsiveness after global ischemia and reflow. Right ventricular trabeculae were dissected from rat hearts subjected either to 20 minutes ischemia and reperfusion in the absence of drugs (stunned group) or to the same protocol in the presence of allopurinol, an inhibitor of xanthine oxidase (XO), and mercaptopropionylglycine (MPG), a hydroxyl radical scavenger (antioxidant group). At 20 minutes of reflow, isovolumic developed pressure recovered completely in the antioxidant group, but in the stunned group it recovered by only 57%. [Ca²⁺]_i and contractile force measurements in trabeculae revealed the expected depression of myofilament function in the stunned group, with no change in Ca²⁺ transients relative to nonischemic controls. In contrast, Ca²⁺ transients were smaller, but force was greater, in the antioxidant group relative to both the stunned group and to nonischemic controls. Steady-state [Ca²⁺]_i-force relationships revealed a striking increase of maximal force and a modest shift of activation to a lower range of [Ca²⁺]_i. The increase in maximal force was reproduced by allopurinol+MPG or by allopurinol alone under nonischemic conditions and also by oxypurinol (100 µmol/L), a potent inhibitor of XO. We conclude that allopurinol and oxypurinol sensitize the cardiac myofilaments to Ca²⁺. This Ca²⁺-sensitizing effect underlies the preservation of contractility observed with an allopurinol+MPG antioxidant cocktail in a model of stunned myocardium. These serendipitous findings identify allopurinol and oxypurinol as the lead compounds of a novel class of inotropic agents.

Key Words: allopurinol • excitation-contraction coupling • ischemia • reperfusion • stunning

	Introduction

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It is generally accepted that the generation of oxygen-derived free radicals (OFRs) during reperfusion after brief ischemia leads to the reversible postischemic myocardial dysfunction known as "stunning."¹ Several lines of investigation have demonstrated that preventing OFR accumulation by antioxidant treatment strongly attenuates stunning (see Reference 1¹ for review). Conversely, exposure of heart muscle to exogenously generated free radicals reproduces many (but not all) of the phenotypic features of stunned myocardium.² The fundamental injury produced by excitation-contraction coupling in stunned myocardium is a decrease of myofilament responsiveness to Ca²⁺ (References 3³ to 6). Therefore, we sought to determine whether antioxidant treatment prevents stunning by preservation of myofilament Ca²⁺ responsiveness after global ischemia and reflow.

In the present study, we used a combination of allopurinol, a xanthine oxidase (XO) inhibitor, and N-2-mercaptopropionylglycine (MPG), a scavenger of hydroxyl anions, to avoid OFR accumulation during ischemia/reperfusion in the rat myocardium. These drugs confer powerful protection against myocardial stunning,^{7 8 9} a finding that we confirmed. Characterization of excitation/contraction coupling revealed not only a striking preservation of the myofilament responsiveness to Ca²⁺ but also an unanticipated blunting of Ca²⁺ transients in the antioxidant group. Even under nonischemic conditions, allopurinol or oxypurinol (a more potent inhibitor of XO)¹⁰ was found to have a striking myofilament-sensitizing effect. The unexpected finding that XO inhibitors are potent myofilament Ca²⁺ sensitizers complicates the interpretation of their protective effect against stunning. A preliminary report has appeared.¹¹

	Materials and Methods

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Whole Rat Hearts
Male rats (LBN-F1 strain, 200 to 250 g, Harlan, Indianapolis, Ind) were anesthetized by intra-abdominal injection of 0.2 to 0.3 mL of sodium pentobarbital (6 grains/mL, ANPRO Pharmaceutical). According to previously described methodology,⁶ the heart was excised and retrogradely perfused at a constant rate of 15 mL/min, with Krebs-Henseleit (K-H) solution equilibrated with a mixture of 95% O₂/5% CO₂. The K-H solution was composed of (mmol/L) NaCl 120, NaHCO₃ 20, KCl 5, MgSO₄ 1.2, glucose 10, and CaCl₂ 1.2, pH 7.35 to 7.40. The hearts were paced at 4.5 Hz, except for a period indicated below, via 2 wire electrodes placed on the right ventricle. Isovolumic left ventricular pressure was measured with an intracavitary balloon filled with water and connected to a pressure transducer. The volume of the balloon was adjusted to a diastolic pressure of 10 mm Hg, which was kept constant for the whole experiment. The heart was placed in a water-jacketed container to maintain a constant temperature of 37°C. Temperature was monitored throughout the experiment by a probe inside the left ventricle. All hearts were initially perfused for 10 minutes to allow stabilization of pressure development and then were subjected to one of the following protocols: (1) In the stunned group, the hearts were perfused for another period of 10 minutes and then subjected to 20 minutes of no-flow global ischemia, followed by 20 minutes of reperfusion. (2) In the antioxidant group, the perfusion protocol was the same as the previous one but in the presence of 0.5 mmol/L allopurinol (Sigma Chemical Co.) and 2.0 mmol/L MPG (Sigma Chemical Co), a hydroxyl radical scavenger, to prevent ·O₂^- and ·OH accumulation, respectively. Both drugs were dissolved directly in the K-H solution after the first 10 minutes of perfusion and remained present throughout the reperfusion period. (3) In the control group, the hearts were perfused continuously for 50 minutes with no drugs. (4) The nonischemic antioxidant group was subjected to 50 minutes of perfusion in the presence of 0.5 mmol/L allopurinol+2.0 mmol/L MPG. (5) The nonischemic oxypurinol group was subjected to 50 minutes of perfusion in the presence of 100 µmol/L oxypurinol. For protocols 1 and 2, pacing was stopped after 3 minutes of ischemia and resumed after 3 minutes of reperfusion. Note that whenever drugs were administered (ie, in groups 2, 4, and 5), they were present only during the perfusion phases in the intact hearts; antioxidants were not added to the muscles after dissection.

Rat Trabeculae
At the end of each protocol, the hearts were removed from the perfusion apparatus and subsequently perfused with a high-K⁺ (20 mmol/L) K-H solution in a dissection dish at room temperature (21°C to 22°C). Trabeculae from the right ventricle of these hearts were dissected and mounted between a force transducer and a micromanipulator in a perfusion chamber placed on the stage of an inverted microscope according to methods already described.^{12 13} The dimensions of the trabeculae were (mm) 2.7±0.1 long, 0.176±.09 wide, and 0.087±0.01 thick (mean±SE, n=22). The cross-sectional area was calculated by multiplying thickness and width and was corrected by a factor of 0.75, assuming an ellipsoidal shape, and a reduction in thickness of 5% because of the stretch to a sarcomere length of 2.2 µm. All muscles were superfused with K-H solution with 0.5 mmol/L CaCl₂ at a flow rate of 10 mL/min and stimulated at 0.5 Hz. All experiments were performed at room temperature. Force was measured as described previously^{12 13} by a silicon strain gauge (model AEM 801, SensoNor) and expressed as mN/mm² of cross-sectional area. All experiments were carried out at the length at which the muscles developed maximal twitch force (end-diastolic sarcomere length of 2.2 to 2.3 µm).

Measurement of [Ca²⁺]_i in Trabeculae
[Ca²⁺]_i was measured with fura-2, according to the method described previously by Backx and ter Keurs.¹⁴ Briefly, after 40 to 60 minutes of stabilization at 0.5 Hz of stimulation frequency, pacing was stopped, and fura-2 pentapotassium salt was microinjected iontophoretically into one cell and allowed to spread throughout the muscle via gap junctions. After fura-2 loading, stimulation was resumed, and [Ca²⁺]_i was determined by epifluorescent illumination at 380 and 340 nm. The fluorescence was collected at 510 nm by a photomultiplier tube (R1527, Hamamatsu). The output of the photomultiplier was filtered at 100 Hz, collected by an analog-digital converter, and stored in a computer for later analysis. [Ca²⁺]_i was calculated by the following equation after subtraction of the corresponding background fluorescence of the trabeculae: [Ca²⁺]_i=K'_d(R-R_min)/(R_max-R), where R is the observed ratio of fluorescence (340/380), K'_d is the apparent dissociation constant, R_max is the ratio 340/380 nm at saturating [Ca²⁺], and R_min is the ratio 340/380 nm at zero [Ca²⁺]. The values of K'_d, R_max, and R_min were 2.95, 9.55, and 0.50, respectively, as determined by in vivo calibrations in the muscles.^{2 6 12 14} The apparent K_d is the result of multiplying the true K_d of fura-2 by a correction factor obtained from the ratio of fluorescence of the Ca²⁺-free to Ca²⁺-bound forms of fura-2 at 380 nm (Sf2/Sb2; for more details see Reference 15¹⁵ ).

Experimental Protocols
To characterize excitation-contraction coupling, we used the following conventional experimental protocols.^{2 6} We first studied the response to [Ca²⁺]_o (0.5, 1.0, 1.5, and 2.0 mmol/L) during twitch contractions elicited by field stimulation (pulse duration, 5 milliseconds) at a rate of 0.5 Hz. Thereafter, the muscles were exposed to 5 µmol/L ryanodine for 30 minutes and stimulated periodically (1 min^-1) at 10 Hz to elicit tetani of 4- to 5-second duration. We varied [Ca²⁺]_o to achieve different levels of steady-state activation during tetani until maximal force was reached.

Steady-state [Ca²⁺]_i-force relationships 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.^{2 6}

In a separate group of experiments (not included above, since we did not perfuse the heart but simply dissected trabeculae immediately after excision), we added 0.5 mmol/L allopurinol directly to the muscle and studied steady-state activations (n=3).

Statistics
Student t test or one-way ANOVA was used for statistical analysis of the data as appropriate.^{16 17} A value of P<0.05 was considered to indicate significant differences between groups. Data are expressed as mean±SE, unless otherwise indicated.

	Results

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Functional Recovery of Isovolumic Left Ventricular Pressure in the Stunned Versus the Antioxidant Group
The starting point for the present study was to determine whether the allopurinol+MPG antioxidant cocktail protects against stunning. The protocol for myocardial stunning was similar to that used in previous work^{4 6} and consisted of 20 minutes of total global ischemia followed by 20 minutes of reflow. We analyzed the functional recovery of the hearts in the absence (stunned group, n=4) and in the presence (antioxidant group, n=5) of the cocktail. Figure 1 shows the averaged left ventricular pressure before and after ischemia (left and right panels), and the percent recovery of left ventricular developed pressure after 20 minutes of reflow in both groups (bar graphs, middle panel). At the end of the reperfusion period, isovolumic developed pressure had recovered completely in the antioxidant group (100±6%), but in the stunned group it recovered by only 57±3% (P<0.05), revealing a full protection against stunning by the antioxidant cocktail. After ischemia, diastolic pressure remained elevated in the antioxidant group. Factors extrinsic to excitation-contraction coupling (such as postischemic edema and vascular turgor) are likely to account for much of this residual increase in diastolic pressure, given that stunned hearts exhibit a large increase of diastolic pressure but very little active force generation at end diastole.⁶

View larger version (14K): [in this window] [in a new window]   Figure 1. Antioxidants improve functional recovery after ischemia in intact hearts. Left and right, Pooled data for peak systolic (triangles) and end-diastolic (circles) isovolumic left ventricular developed pressure (LVDP) in stunned (left) and antioxidant (right) groups. Middle, Bar graph summarizing percent recovery of LVDP (ie, systolic minus diastolic pressure) in the 2 groups (solid bar, stunned group; open bar, antioxidant group). All. indicates allopurinol. *P<0.05 between the 2 groups.

Force-[Ca²⁺]_i Relationships During Twitch Contractions in Stunned Versus Antioxidant Group Trabeculae
Trabeculae from both groups of hearts were dissected and mounted between a force transducer and a micromanipulator in a perfusion chamber on the stage of an inverted microscope. When the muscles were homogeneously loaded with fura-2, we began the characterization of Ca²⁺ transients and the corresponding force development. Figure 2 shows Ca²⁺ transients and force in typical experiments from the stunned and antioxidant group. The top panels show that Ca²⁺ transients were smaller in amplitude in the muscle from the antioxidant group. Nevertheless, the force generated by this muscle was considerably greater. The pooled data for [Ca²⁺]_i (top) and force (bottom) in Figure 3 illustrate the consistency of these findings. Regardless of the [Ca²⁺]_o studied, [Ca²⁺]_i was lower, and force was higher, in muscles from the antioxidant group (although the difference in force did not reach statistical significance at 1.5 and 2.0 mmol/L [Ca²⁺]_o). The finding that the antioxidant muscles generate more force with smaller Ca²⁺ transients signifies that their myofilaments are sensitized to [Ca²⁺]_i relative to the stunned group. If that were true, one might predict an increase in diastolic force without a net increase in diastolic [Ca²⁺]_i. Figure 4 shows this was indeed the case; although the end-diastolic [Ca²⁺]_i was not different in the 2 groups, the end-diastolic force was significantly higher in the antioxidant group at any given [Ca²⁺]_o. We also examined the rates of relaxation by quantifying the half-times of decay of force and [Ca²⁺]_i; these did not differ significantly in the antioxidant and stunned groups (data not shown).

View larger version (21K): [in this window] [in a new window]   Figure 2. Ca2+ transients and force during twitch contractions in representative stunned and antioxidant muscles. [Ca2+]i (top) and force (bottom) are shown in trabeculae dissected from a heart from the stunned group (left) and from the antioxidant group (right), at [Ca2+]o of 0.5 mmol/L.

View larger version (18K): [in this window] [in a new window]   Figure 3. Systolic [Ca2+]i and force at various [Ca2+]o levels. Pooled data are shown for peak systolic [Ca2+]i (top) and peak systolic force (bottom) in the stunned (solid bars) and antioxidant (open bars) groups. *P<0.05 between the 2 groups.

View larger version (14K): [in this window] [in a new window]   Figure 4. Diastolic [Ca2+]i and force at various [Ca2+]o levels. Pooled data are shown for end-diastolic [Ca2+]i (top) and end-diastolic force (bottom) in the stunned (solid bars) and antioxidant (open bars) groups. *P<0.05 between the 2 groups.

These results suggest that the muscles in the antioxidant group had a preserved myofilament responsiveness to Ca²⁺, but we cannot ignore the fact that the Ca²⁺ transients were blunted in the antioxidant group. In fact, these Ca²⁺ transients are smaller than not only those in the stunned group but also those in numerous nonischemic control groups reported earlier.^{2 6 12 13} This finding is inconsistent with a simple preservation of myofilament Ca²⁺ responsiveness. Instead, the results suggest that the antioxidant cocktail itself has a primary myofilament Ca²⁺-sensitizing effect; this possibility will be addressed later.

Steady-State [Ca²⁺]_i-Force Relationships in Stunned Versus Antioxidant Group Trabeculae
Although the results shown in Figures 2 to 4 indicate an augmented myofilament Ca²⁺ responsiveness in the antioxidant group, steady-state analysis is required to determine whether this is a manifestation of increased F_max and/or enhanced myofilament Ca²⁺ sensitivity. We thus performed steady-state activations of the muscles by tetanization.^{2 6 12} Figure 5 shows representative records of [Ca²⁺]_i and force during tetanic activation in muscles from the stunned (A) and antioxidant (B) groups (10 mmol/L [Ca²⁺]_o). Despite the similar [Ca²⁺]_i reached during both activations, the stunned group developed significantly lower force than did the antioxidant group. Pooled data for the steady-state activations obtained at different [Ca²⁺]_o levels for all muscles in the stunned group (n=4) and in the antioxidant group (n=5) are shown Figure 5C. All points falling within 250 nmol/L–wide bins of [Ca²⁺]_i were pooled to produce the data shown for [Ca²⁺]_i and force. To facilitate comparison between experimental groups, experimental curves in each group were normalized and then scaled by the averaged maximal force in that experimental group.^{2 6 12} Thus, each curve plots the average absolute maximal force at saturating [Ca²⁺]_i in the corresponding experimental group; other data within each group are scaled relative to that group's mean value. The antioxidant treatment clearly protected against stunning, since the steady-state [Ca²⁺]_i-force relationships revealed a striking preservation of maximal force (135±17 versus 56±10 mN/mm² for antioxidant treatment versus stunning, respectively; P<0.05). The curve also shifted slightly to the left (ie, to lower [Ca²⁺]_i), but the [Ca²⁺]_i required to activate 50% of the maximal force was not significantly different in the 2 groups (K_d, 0.831±0.106 versus 0.633±0.095 µmol/L for stunning versus antioxidant treatment, respectively; P=NS). Thus, the major effect was an increase of F_max.

View larger version (16K): [in this window] [in a new window]   Figure 5. Steady-state contractile activation in stunned and antioxidant groups. Tetani were stimulated in individual muscles from each group at various [Ca2+]o levels, and the steady-state values of [Ca2+]i and force were quantified. Panels A and B show typical records of [Ca2+]i and force in muscles from the stunned (A) and antioxidant (B) groups at 10 mmol/L [Ca2+]o. In panel C, pooled data represent mean±SE of all data points obtained during steady-state activations at different [Ca2+]o levels in each group, which fell within bins of 250 nmol/L width (eg, 0 to 249 nmol/L, 250 to 299 nmol/L). Mean absolute maximal force is shown in each group, and the pooled data in each group were scaled accordingly.

Are the results simply due to preserved sensitivity of the myofilaments to Ca²⁺, or could there be a primary effect of the antioxidant cocktail on the myofilaments? To consider this possibility further, we compared steady-state activation in the antioxidant group to that in nonischemic controls. Figure 6 shows that, despite having been subjected to 20 minutes of ischemia and reflow, muscles from the antioxidant group tended to achieve higher levels of maximal force than did the muscles from the control group (135±17 versus 111±8 mN/mm², P=NS). There would be no reason to expect even a small net increase in maximal force in the antioxidant group relative to nonischemic controls if the only mechanism of protection were simple mitigation of OFR effects on reflow. Therefore, we next examined the effects of antioxidants in the absence of superimposed ischemia and reflow.

View larger version (15K): [in this window] [in a new window]   Figure 6. Steady-state contractile activation in control and antioxidant groups. Data were analyzed and plotted as described in the legend to Figure 5.

Force-[Ca²⁺]_i Relationships During Twitch Contractions in Nonischemic Control and Antioxidant Groups
Figure 7 shows Ca²⁺ transients and force records from representative muscles in the control and nonischemic antioxidant groups. The muscle that had been exposed to allopurinol and MPG (right) exhibits a reduced Ca²⁺ transient but enhanced force relative to the control muscle from a heart perfused without drugs (left). These findings were consistent at any given [Ca²⁺]_o, supporting the idea that antioxidants exert a myofilament Ca²⁺-sensitizing effect in the absence of ischemia. To compare the 2 groups further, we fit the peak [Ca²⁺]_i and peak force obtained at 0.5, 1.0, 1.5, and 2.0 mmol/L [Ca²⁺]_o to linear functions,^{6 12} while recognizing that the relationship between peak [Ca²⁺]_i and peak force need not be linear.¹⁸ The slope of this relationship was significantly greater in the antioxidant group (156±41 versus 61±15 mN · mm^-2 · (µmol/L)^-1, P<0.05), revealing a tendency of these muscles to develop more force at any given [Ca²⁺]_i. Once again, we examined the rates of relaxation by quantifying the half-times of decay of force and [Ca²⁺]_i; these did not differ significantly in the control and nonischemic antioxidant groups (data not shown). To distinguish between effects on sensitivity and maximal force, we next determined the steady-state [Ca²⁺]_i-force relationships in the 2 experimental groups.

View larger version (22K): [in this window] [in a new window]   Figure 7. Ca2+ transients and force during twitch contractions in representative control and nonischemic antioxidant muscles. [Ca2+]i (top) and force (bottom) are shown in trabeculae dissected from a control heart (left) and a nonischemic antioxidant heart (right), at [Ca2+]o of 2 mmol/L.

Steady-State [Ca²⁺]_i-Force Relationships in Nonischemic Control Versus Antioxidant Groups
The steady-state data in Figure 8 reveal a sizable increase in maximal force in the nonischemic antioxidant group (152±2 versus 111±8 mN/mm², P<0.05). Interestingly, the increase in Ca²⁺ responsiveness reflects a pure augmentation of maximal force: the midpoint of activation (K_d) did not differ significantly in the 2 groups (0.573±0.116 versus 0.473±0.031 µmol/L, P=NS), nor did the shape or steepness of the curves differ when normalized to the same maximal force (not shown).

View larger version (13K): [in this window] [in a new window]   Figure 8. Steady-state contractile activation in control and nonischemic antioxidant groups. Data were analyzed and plotted as described in the legend to Figure 5.

The results shown above indicate that the combination of an XO inhibitor and a hydroxyl radical scavenger mitigates stunning; this apparent protection in fact reflects primary myofilament Ca²⁺ sensitization by the antioxidants. The effects of allopurinol and MPG revealed above are actually aftereffects, as the muscles had been exposed to the drugs only during perfusion of the hearts (1 hour before the recordings of Ca²⁺ and force in the isolated trabeculae). The fact that the effects are persistent hints that this result is produced mainly by allopurinol by virtue of its action as an XO inhibitor rather than by a free radical–scavenging effect of MPG (which would depend on the continued presence of the drug). To distinguish directly which of the 2 compounds was responsible for the myofilament sensitization, we first applied allopurinol directly to 3 muscles and quantified steady-state activation, and then (in a separate series of experiments) we perfused hearts with oxypurinol, a high-affinity inhibitor of XO (K₁=100 nmol/L, bovine),¹⁹ and once again quantified steady-state activations in trabeculae.

Steady-State [Ca²⁺]_i-Force Relationships After Acute Addition of Allopurinol and After Perfusion With Oxypurinol
Figure 9 compares the mean steady-state [Ca²⁺]_i-force relationships in 3 groups: in control muscles (solid circles), in 3 muscles exposed acutely to 0.5 mmol/L allopurinol (open circles), and in 3 muscles from hearts that had been perfused with 100 µmol/L oxypurinol (open triangles). The results obtained after acute exposure to allopurinol were almost indistinguishable from those observed previously as an aftereffect of allopurinol and MPG combined: the XO inhibitor alone increased maximal force (150±5 versus 111±8 mN/mm², P<0.05), with no change in K_d. This finding confirms that allopurinol has a sensitizing effect on the rat myocardium, possibly mediated by its known XO inhibitory action. To probe further the mechanism of the allopurinol effect, we determined whether oxypurinol (a high-affinity inhibitor of XO¹⁹) also produces myofilament sensitization. Figure 9 shows that oxypurinol significantly increased maximal force (144±5 versus 111±8 mN/mm², P<0.05) to a degree comparable to that observed with the full antioxidant cocktail and with allopurinol alone, once again without affecting the position or the steepness of the steady-state activation curve. These results demonstrate directly the myofilament Ca²⁺-sensitizing property of XO inhibitors.

View larger version (18K): [in this window] [in a new window]   Figure 9. Steady-state contractile activation in control muscles, in 3 muscles acutely exposed to 0.5 mmol/L allopurinol alone, and in 3 muscles from hearts that had been perfused with 100 µmol/L oxypurinol. Data were analyzed and plotted as described in the legend to Figure 5.

	Discussion

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Our findings have 3 major implications: first, for the mechanism of antioxidant protection against reperfusion injury; second, for therapeutics of acute and chronic cardiac pump failure; and third, for the notion that oxidants function as physiological signaling molecules. These concepts will be considered in turn.

Mechanism of Antioxidant Protection Against Stunning
Antioxidants are known to mitigate the postischemic cardiac contractile dysfunction known as stunning. In the present study, we used a combination of allopurinol, an inhibitor of XO, and MPG, a scavenger of ·OH, to avoid OFR accumulation during ischemia/reperfusion in the rat myocardium. We found a strong protection against stunning with this drug combination, as manifested by full recovery of the left ventricular developed pressure after 20 minutes of reperfusion. This was not entirely surprising, since other authors had shown similar results with various combinations of antioxidants.^{1 7 8 9 19 20 21}

The 2 main OFR species implicated in the pathogenesis of myocardial stunning are ·O₂^- (superoxide) and ·OH (hydroxyl radical). Both are produced in the postischemic heart as a burst during early reperfusion, when tissue is suddenly reoxygenated.⁷ The likely sequence of OFR production is as follows: ATP is rapidly consumed during ischemia, and its breakdown products (notably adenosine, inosine, and hypoxanthine) accumulate in the myocardium. XO is a free radical–generating enzyme that uses O₂ as its electron acceptor. Reperfusion introduces molecular oxygen, enabling XO to generate ·O₂^- and xanthine, and in a second round of catalysis, xanthine is broken down to uric acid with the generation of more ·O₂^- (Reference 22²² ). Superoxide dismutase (SOD) then converts ·O₂^- to H₂O₂ in the presence of catalytic iron; finally, ·O₂^- and H₂O₂ interreact in a Haber-Weiss reaction to generate the highly reactive ·OH (Reference 23²³ ). H₂O₂ can also produce ·OH through a Fenton reaction.^{1 19 23} Although evidence from intact animal models indicates that both ·O₂^- and ·OH are involved in stunning,¹ the primary methodological implications of the present study relate to the use of interventions targeted at ·O₂^-. Studies using inhibitors of XO have been interpreted as reflecting a simple inhibition of ·O₂^- accumulation during reflow; the possibility that such compounds alter excitation-contraction coupling has been overlooked.

Our results indicate that the XO inhibitors allopurinol and oxypurinol have profound and unanticipated effects on the Ca²⁺ responsiveness of the myofilaments. Because both XO inhibitors have similar phenotypic consequences, we have provisionally attributed our findings to the inhibition of XO. The observations that both compounds produce a persistent increase in myofilament responsiveness long after washout are also consistent with their ability, as pseudosubstrates for XO, to block the enzyme for long periods. It is unclear precisely how blockage of XO might produce the changes in myofilament responsiveness, although one obvious possibility is inhibition of basal production of ·O₂^-. This idea is discussed more extensively below. Nevertheless, we cannot exclude an unrelated, and previously unknown, action of these compounds on the myofilaments. In any case, the present results complicate the interpretation of previous ischemia/reperfusion studies with XO inhibitors. The finding of improved functional recovery clearly does not reflect a mere inhibition of the ·O₂^- burst on reflow. Likewise, if the effects turn out to reflect suppression of basal ·O₂^- production, similar cautions may apply to interpretation of the beneficial effects of SOD and SOD-mimetic compounds.

We have previously argued that partial proteolysis of the thin-filament regulatory protein troponin I underlies the contractile dysfunction of stunned myocardium.²⁴ The mitigation of stunning seen here as a consequence of antioxidant treatment might conceivably involve a reduction of the amount of troponin I proteolysis; we have not tested this prediction. However, such a reduction of proteolysis is unlikely to account for the potentiation of maximal force in nonischemic myocardium, given the absence of any detectable troponin I degradation in the basal state.^{24 25 26} Numerous known interactions among kinase/phosphatase systems, NO metabolism, and oxidant pathways^{27 28} indicate that these aspects of signal transduction merit further investigation as possible contributors to the salutary action of antioxidants on the myofilaments.

Implications for Therapeutics
Ca²⁺ sensitizers have been proposed as novel therapeutic agents for heart failure and other disorders of contractility. By acting on the final step of excitation-contraction coupling, such agents have the potential to improve function without directly altering Ca²⁺ cycling or signal transduction pathways.²⁹ A variety of pharmaceuticals act as Ca²⁺ sensitizers, but the existing ones suffer from several shortcomings. These include the generic concern that drugs that shift Ca²⁺ sensitivity, causing force to be activated at lower-than-normal [Ca²⁺]_i, could impair diastolic relaxation.³⁰ Many of the presently available drugs are also phosphodiesterase inhibitors³¹ and thus have the undesirable feature of increasing intracellular cAMP concentration.³²

As novel Ca²⁺ sensitizers, allopurinol and oxypurinol offer several potential advantages: (1) These agents differ chemically from known sensitizers (although there is some structural kinship to the imidazole-containing "natural sensitizers"³³) and thus serve as the lead compounds for a new class of therapeutic agents. (2) Unlike other known sensitizers, the XO inhibitors purely increase maximal force, without shifting the range of contractile activation to lower [Ca²⁺]_i. In principle, crossbridge kinetics could be optimized so as to enhance the fraction of active crossbridges during each cardiac cycle.³⁰ Agents with such a Ca²⁺-sensitizer action may improve the economy of cardiac contraction without impairing relaxation. Allopurinol and oxypurinol appear to be the first Ca²⁺-sensitizing drugs that purely increase maximal force and thus may offer unique advantages over existing compounds. (3) The sensitizing effect was proportionally greater in postischemic myocardium (>2-fold increase of F_max, Figure 5) than in nonischemic myocardium (40% increase of F_max, Figures 8 and 9). This observation indicates that allopurinol exerts a greater sensitizing effect on myofilaments whose responsiveness is blunted at baseline. Myofilament Ca²⁺ responsiveness is blunted not only in the stunned myocardium but also during ischemia and hypoxia and possibly in chronic heart failure,³⁴ giving reason to predict that the XO inhibitors may preferentially boost contractility in these disease states. (4) The increase in force occurs despite a decrease in Ca²⁺ transient amplitude. We have not yet investigated the mechanism of the decrease in [Ca²⁺]_i, but it may reflect enhanced Ca²⁺ binding to the myofilaments, as has been seen with other sensitizers (eg, sulmazole³⁵ ). Such an action would not be expected with pure class II sensitizers, unless there is some feedback between force generation and Ca²⁺ binding.³⁶ In any case, diminution of [Ca²⁺]_i, whether primary or secondary, may itself represent a useful therapeutic principle given the importance of cellular Ca²⁺ overload in a variety of pathologies.³⁷ (5) Both XO inhibitors have been in clinical use for decades (allopurinol for gout and oxypurinol as its active metabolite), so that the safety of these compounds is well established.

The major limitation in applying XO inhibitors to humans with cardiac pump failure may turn out to be a relative paucity of this enzyme in human heart. The literature on species differences in XO expression is extensive but contradictory. For example, in the rat heart, McCord et al²⁰ argued that XO is produced only during ischemia by a proteolytic conversion of xanthine dehydrogenase, whereas other authors have shown significant amounts of the enzyme under nonischemic conditions.^{19 21} Human heart has long been thought to exhibit very low, if any, XO activity,^{38 39} but more recent studies have demonstrated substantial quantities of the enzyme.^{40 41 42} The possibility that expression of the enzyme is increased in heart failure is particularly relevant and is substantiated by the observation of hyperuricemia in patients with decompensated valvular heart disease.⁴³ Of course, the quantity of XO will be irrelevant if the mechanism of sensitization turns out to be a direct effect unrelated to inhibition of the enzyme.

Superoxide as a Physiological Signaling Molecule
The broadest biological implications of the present study revolve around the possibility that the XO inhibitors augment myofilament sensitivity by blocking the basal production of ·O₂^-. Perhaps tonic levels of ·O₂^- in normal heart suffice to decrease myofilament sensitivity, a directional effect consistent with reports that exogenously generated ·O₂^- depresses the Ca²⁺ responsiveness of the contractile proteins.^{2 44} The physiological regulation of the myofilaments in intact muscle is still poorly understood, but it is increasingly clear that Ca²⁺ responsiveness is far from static. Superoxide, as a byproduct of energy metabolism, would help to make contractility responsive to the energetic state of the muscle. Until recently, ·O₂^- was regarded purely as a toxic substance. Nevertheless, the finding that reactive oxygen species (probably ·O₂^-) act as second messengers for growth factors in fibroblasts⁴⁵ supports the general idea that OFRs might be involved in physiological signaling.

	Acknowledgments

 
This study was supported by NIH grant RO1 HL-44065 and by a fellowship from the CONICET of Argentina (Dr Pérez).

	Footnotes

 
This manuscript was sent to Harry A. Fozzard, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Presented as preliminary results in abstract form (Circulation. 1997;96[suppl I]:I-200).

Received February 18, 1998; accepted May 6, 1998.

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