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(Circulation Research. 2000;86:1211.)
© 2000 American Heart Association, Inc.

Integrative Physiology

2-Deoxy-ATP Enhances Contractility of Rat Cardiac Muscle

M. Regnier, A. J. Rivera, Y. Chen, P. B. Chase

From the Departments of Bioengineering (M.R., A.J.R.), Radiology (Y.C., P.B.C.), and Physiology and Biophysics (P.B.C.), School of Medicine, University of Washington, Seattle, Wash.

Correspondence to Michael Regnier, Department of Bioengineering, Box 357962, School of Medicine, University of Washington, Seattle, WA 98195-7962. E-mail mregnier{at}u.washington.edu

	Abstract

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Abstract—To investigate the kinetic parameters of the crossbridge cycle that regulate force and shortening in cardiac muscle, we compared the mechanical properties of cardiac trabeculae with either ATP or 2-deoxy-ATP (dATP) as the substrate for contraction. Comparisons were made in trabeculae from untreated rats (predominantly V1 myosin) and those treated with propylthiouracil (PTU; V3 myosin). Steady-state hydrolytic activity of cardiac heavy meromyosin (HMM) showed that PTU treatment resulted in >40% reduction of ATPase activity. dATPase activity was >50% elevated above ATPase activity in HMM from both untreated and PTU-treated rats. V_max of actin-activated hydrolytic activity was also >50% greater with dATP, whereas the K_m for dATP was similar to that for ATP. This indicates that dATP increased the rate of crossbridge cycling in cardiac muscle. Increases in hydrolytic activity were paralleled by increases of 30% to 80% in isometric force (F_max), rate of tension redevelopment (k_tr), and unloaded shortening velocity (V_u) in trabeculae from both untreated and PTU-treated rats (at maximal Ca²⁺ activation), and F-actin sliding speed in an in vitro motility assay (V_f). These results contrast with the effect of dATP in rabbit psoas and soleus fibers, where F_max is unchanged even though k_tr, V_u, and V_f are increased. The substantial enhancement of mechanical performance with dATP in cardiac muscle suggests that it may be a better substrate for contractility than ATP and warrants exploration of ribonucleotide reductase as a target for therapy in heart failure.

Key Words: in vitro motility • hypothyroid • myosin isoforms • contractile kinetics • shortening velocity

	Introduction

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In both cardiac and skeletal muscles, contraction results from the cyclic interactions between actin and myosin when chemical energy, supplied by hydrolysis of MgATP, is converted into mechanical work, force, and shortening (chemomechanical transduction). Skeletal myosin and actomyosin ATPase activity have been studied extensively in solution, and a detailed picture of the number of steps and rate-limiting processes of the actomyosin ATPase cycle now exists (reviewed in Reference ¹ ). More recently, similar analyses have been conducted in chemically skinned, fast skeletal muscle fibers using measurements of force transients that follow quick length changes; a variety of strong crossbridge inhibitors; or the photolysis of caged compounds such as ATP, ADP, P_i, and Ca²⁺ (reviewed in Reference ² ). These studies are necessary to elucidate the chemomechanical transduction pathway, because the orderly array of thin and thick filaments in muscle imposes constraints on actomyosin interactions. The results of these studies have led to detailed models of chemomechanical transduction that suggest the rate-limiting processes controlling steady-state force and the rate of force production and shortening (reviewed in Reference ² ). Much less work has been done to determine the processes controlling force production and shortening in cardiac muscle, although these are often assumed to be the same as for skeletal muscle. Although evidence from solution measurements suggests that the basic ATPase pathway in cardiac muscle is similar to that in skeletal muscle,^{3 4 5} a detailed study of the chemomechanical cycle in skinned cardiac muscle is needed.

One particularly fruitful method of studying chemomechanical transduction in skeletal muscle has been to substitute analogs of ATP (NTPs) as the substrate for contraction.^{6 7 8 9 10 11} Correlation of the mechanical behavior using NTPs with changes in rate constants of actomyosin "state" transitions can provide useful information about mechanically and energetically important steps during the hydrolysis cycle. Recently, Regnier et al^{10 11} used a series of naturally occurring nucleotide analogs of ATP (NTPs) to study the kinetic regulation of the chemomechanical cycle in rabbit psoas muscle fibers. Most of these analogs bind more slowly to myosin, support less force and shortening, and have slower steady-state hydrolysis rates than when MgATP is the substrate for contraction. However, one of these analogs, 2-deoxy-ATP (dATP), uniquely had an affinity for myosin similar to that of ATP and increased both solution NTPase and rate of crossbridge cycling in psoas fibers. The rate of force development and fiber shortening velocity were also moderately increased by dATP with no effect on isometric force of muscle fibers during maximal Ca²⁺ activation. Characterization of the posthydrolysis steps that control steady-state force and the kinetics of force generation in fibers led to the conclusion that dATP increases transition rates at both the beginning and the end of the crossbridge power stroke, resulting in a faster crossbridge cycling rate with no increase in number of strongly attached crossbridges contributing to steady-state force.

Because dATP is an effective substrate for fast skeletal myosin and is also rapidly rephosphorylated from dADP to dATP by creatine phosphokinase,¹⁰ we chose to compare ATP with dATP as the contractile substrate to begin our studies of chemomechanical transduction in cardiac muscle. In this study, we compared ATP with dATP for cardiac trabeculae contractions, for solution NTPase measurements, and for in vitro motility studies with purified F-actin and HMM. Surprisingly, we found that dATP substantially increases the level of steady-state force and stiffness in skinned cardiac trabeculae containing either V1 or V3 myosin isoforms. This contrasts with our previous results in fast skeletal muscle and our current results in slow skeletal muscle. Further study showed that both the rate of tension redevelopment (k_tr) and the rate of crossbridge cycling in cardiac muscle are increased by dATP to a greater extent than in fast skeletal muscle fibers. Our results indicate that dATP may be a better contractile substrate than ATP in cardiac muscle and that the processes controlling maximal Ca²⁺-activated force development and shortening in cardiac muscle may differ from those in fast skeletal muscle. Preliminary reports of this work were published previously.^{12 13}

	Materials and Methods

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Mechanical Experiments
Male Sprague-Dawley rats (200 to 250 grams), either treated with propylthiouracil (PTU; 0.8% added to drinking water for 4 to 8 weeks) or untreated (as age-matched animals) were euthanized with sodium pentobarbital (50 mg/kg). Animal care and handling conformed to the National Institutes of Health policy on humane care and use of laboratory animals. Procedures were approved by the University of Washington Animal Care Committee. Hearts were excised, and the interior wall of the right ventricle was exposed to relaxing solution (see below) containing glycerol (50% vol:vol) and Triton X-100 (1%) overnight at 5°C. Individual trabeculae were dissected and stored at 5°C for up to 4 days. Glycerinated rabbit psoas and soleus fibers were prepared as previously described.¹⁴

Trabeculae and skeletal fiber ends were wrapped in aluminum foil T-clips for attachment to a force transducer (either model 400A, 2.2-kHz resonant frequency [Cambridge Technology], or model AE801, 5-kHz resonant frequency [SensoNor]) and a servo-motor (model 300, Cambridge Technology) tuned for a 300-µs step response. Trabecular sarcomere length (L_s) was measured with helium-neon laser diffraction¹⁵ and set to 2.25 µm at pCa 9.2; skeletal fiber L_s was set to 2.55 µm. The following measurements were made as previously described: stiffness determined by sinusoidal length oscillations (500 Hz and 1000 Hz) and steady-state isometric force,¹⁵ rate of isometric tension redevelopment (k_tr),¹⁶ and unloaded shortening velocity (V_u) using the "slack-test" method.^{15 16 17}

Solutions contained (in mmol/L) phosphocreatine 15, EGTA 15, MOPS at least 40, free Mg²⁺ 1, Na⁺ plus K⁺ 135, and DTT 1, and 250 U/mL creatine kinase (CK, Sigma), as well as either 5 mmol/L ATP or 5 mmol/L dATP (Sigma) at pH 7.0 and 15±1°C. Ionic strength was 0.2 mol/L. Affinity of dATP and ATP for Mg²⁺ was assumed to be the same.¹⁶ For activation solutions, the Ca²⁺ level (expressed as pCa =-log [Ca²⁺]) was set to pCa 4.5 or pCa 4.0 by adjusting Ca(propionate)₂.

Solution Assays
Cardiac myosin was prepared from untreated and PTU-treated rat hearts by modification of previously reported methods.^{18 19} Heavy meromyosin (HMM) was obtained by 1-chloro-3-tosylamido-7-amino-2-heptanone–treated chymotryptic digestion of freshly prepared cardiac myosin,²⁰ stored on ice, and used within 2 days. Skeletal muscle myosin and HMM were prepared from rabbit back muscle as previously described.^{21 22} F-actin was prepared from rabbit back and leg muscle ether powder.^{22 23}

ATPase and dATPase activities (NTPase) of cardiac HMM were measured at 21°C to 23°C using a colorimetric method as described.^{21 24} Maximum hydrolysis rate (V_max) and K_m were estimated from increasing F-actin concentrations ([A]=0 to 25 µmol/L) for a single HMM preparation, using a hyperbolic fit to the data {y=V_max [A]/([A]+K_m)} (Figure 1).

View larger version (13K): [in this window] [in a new window]   Figure 1. F-actin concentration dependence of acto-HMM NTPase for muscle from untreated rat hearts at 22°C. Reaction conditions were as described in Materials and Methods with 1.0 and 0.1 mg · mL-1 HMM for HMM alone and acto-HMM NTPase assays, respectively. Individual data points are mean±SE of linear regression fits (r20.97) to Pi accumulation for 5 time points (0 to 10 minutes for acto-HMM and 0 to 60 minutes for HMM alone). NTPase was significantly (P0.01) elevated by dATP (over ATP) for each concentration of F-actin, [A]. Values for Vmax and Km (Table 1) were obtained by fitting these data (solid lines) to the equation y=Vmax([A]/([A]+Km).

In Vitro Motility
Assays were carried out at 30°C using unregulated F-actin and analyzed as described,^{22 24 25 26} except that flow cells contained cardiac HMM from untreated or PTU-treated rats. The [NTP] was varied from 0.003 to 3 mmol/L to determine maximal V_f (V_f(*) (ie, V_f at saturating NTP) and K_app (ie, [NTP] to produce 0.5 V_f(*)) from the equation y=V_f(*) [NTP]/([NTP]+K_app).

An expanded Materials and Methods section is available online at http://www.circresaha.org.

	Results

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Solution NTPase Activity
The steady-state hydrolytic activity (V_HMM) of cardiac HMM from PTU-treated rats was 40% lower than that from untreated rats (Table 1). PTU treatment induces hypothyroidism, which causes myosin heavy chain (MHC) switching in the heart from predominantly -MHC (V1) to ß-MHC (V3).^{27 28 29 30} For both preparations, substituting ATP with dATP increased V_HMM 65%. The rate of NTP hydrolysis by HMM was increased by F-actin and displayed saturation kinetics at high actin concentrations, allowing estimates of K_m and V_max (Table 1). The V_max (actin-activated) of normal HMM was increased >80-fold over V_HMM (without actin) for both ATP and dATP, with a similar K_m for F-actin concentration (10 µmol/L), and dATP increased V_max by 53%. The increases in V_HMM and V_max, and similar K_m for actin, resemble the effects of dATP on fast skeletal HMM.¹⁰

View this table: [in this window] [in a new window]   Table 1. Steady-State MgNTPase Rate of HMM in the Absence and Presence of F-Actin

Mechanical Experiments
To determine how changing the hydrolytic activity of myosin influences the mechanical properties of cardiac muscle and to compare with our previous studies with fast skeletal muscle fibers, we measured maximum Ca²⁺-activated (pCa 4.0) steady-state force (F_max), the rate of tension redevelopment (k_tr), and the rate of unloaded shortening (V_u) in cardiac trabeculae from normal and PTU-treated rats. In chemically skinned trabeculae, F_max was increased substantially with 5 mmol/L dATP compared with 5 mmol/L ATP. Figure 2A shows an example force record for a trabecula in which the contractile substrate was alternated between ATP and dATP. The trabecula was initially activated with ATP until a steady-state level of force was achieved; it was then transferred to a similar solution containing dATP, and force began to rise immediately. Once force leveled out, the trabecula was returned to the ATP solution, then again to the dATP, before being relaxed (pCa 9.2). This activation sequence demonstrates that the substantial increase in F_max with dATP was rapidly and completely reversible. The increase in F_max with dATP was consistent between preparations and occurred even when trabeculae were relaxed subsequent to activation with ATP. An example of this protocol is shown for a rabbit psoas fiber in Figure 2B and a rabbit soleus fiber in Figure 2C. These force records demonstrate that, in sharp contrast to trabeculae (panel A), dATP causes little or no increase in F_max in fast skeletal fibers, as we have previously reported,^{10 11 16} or in slow skeletal fibers. The results of several experiments are summarized in Table 2. F_max was increased by 41±4% with dATP in trabeculae from untreated rats and by 45±5% in trabeculae from PTU-treated rats. In comparison, there was no increase in F_max with dATP in either psoas or soleus fibers. To determine whether the increase in trabecular F_max with dATP resulted from an increase in the number of strongly bound crossbridges, we measured both force and stiffness in 4 trabeculae from untreated rats. For these trabeculae, F_max was increased by 37±2%, and stiffness was increased proportionally by 35±2%, suggesting that, indeed, increases in F_max resulted from an increase in the number of strongly bound crossbridges. In both psoas fibers¹¹ and soleus fibers, dATP had no effect on stiffness (data not shown), similar to the lack of effect on F_max.

View larger version (39K): [in this window] [in a new window]   Figure 2. Maximal Ca2+-activated force with ATP vs dATP. Force records are for an untreated rat trabecula (A), a rabbit psoas fiber (B), and a rabbit soleus fiber (C), all at 15°C. Contractile substrate (ATP or dATP) is indicated above each trace, and Ca2+ concentrations (pCa) of activation sequences are indicated below each trace. Force transients occur every 5 seconds because of ramp release/restretch cycles. Force augmentation by dATP in this trabecula (70%) was greater than average (Table 2). In contrast to cardiac muscle, force was not augmented by dATP in either psoas or soleus fibers. Scale bars in each panel: 30 mg (y axis) and 30 seconds (x axis).

View this table: [in this window] [in a new window]   Table 2. Effect of dATP on the Mechanical Properties of Rat Cardiac and Rabbit Skeletal Muscle and In Vitro F-Actin Motility

The increase in F_max with dATP in cardiac muscle was accompanied by an increase in k_tr of similar magnitude. The example k_tr traces in Figure 3 show sequential activations in ATP, then dATP for trabeculae from one untreated rat (Figure 3A) and one PTU-treated rat (Figure 3B) and for a soleus fiber (Figure 3C). In these examples, F_max was increased by 41% and 43%, whereas k_tr was increased by 55% and 48% in the trabecula from the untreated and PTU-treated rats, respectively. In comparison, dATP increased F_max by only 5% and approximately doubled k_tr in the soleus fiber. The k_tr data for all experiments are summarized in Table 2. In trabeculae from PTU-treated rats, k_tr was >2-fold slower than in trabeculae from untreated rats. With dATP, k_tr was increased by 52% in trabeculae from untreated rats and by 38% in trabeculae from PTU-treated rats. An increase in k_tr with dATP also occurs in both fast and slow skeletal muscle (without a concomitant increase in F_max), but to a lesser degree in psoas fibers (Table 2).

View larger version (19K): [in this window] [in a new window]   Figure 3. Example force traces showing rate of tension redevelopment (ktr) for trabeculae from an untreated rat (A) and a PTU-treated rat (B) or a rabbit soleus fiber (C), comparing ATP and dATP. Rates indicated next to each force trace demonstrate that dATP increased ktr in all 3 preparations. Preparation diameters were 87 µm (A), 93 µm (B), and 67 µm (C).

To determine whether dATP enhances sarcomere shortening as well as force production in cardiac muscle, we measured the unloaded shortening velocity (V_u) of trabeculae using the slack test (see Materials and Methods). Figure 4 shows "slack" times for 6 different length steps in an example trabecula, comparing activations with ATP versus dATP; the data for 6 trabeculae from untreated rats and 3 trabeculae from PTU-treated rats, with length steps ranging between 7% and 15%, are summarized in Table 2 (r² values for V_u ranged from 0.83 to 0.99). V_u was 3-fold slower for trabeculae from PTU-treated rats compared with trabeculae from untreated rats during activations with ATP, consistent with the difference in solution ATPase activities (Table 1). dATP increased V_u by an average of 74% in trabeculae from untreated rats and >2-fold in trabeculae from PTU-treated rats. These increases in V_u with dATP were greater in magnitude than the increases in F_max and k_tr. The increased V_u with dATP in cardiac muscle was similar to or greater than the effect of dATP in soleus fibers and at least 2-fold greater than the effect of dATP in fast skeletal muscle (Table 2).¹⁶

View larger version (14K): [in this window] [in a new window]   Figure 4. Measurement of unloaded shortening velocity (Vu) in trabeculae. For this example, slack times (see Materials and Methods) for 6 length steps, ranging from 7% to 12% of Lo, are shown for maximum Ca2+ activation with ATP vs dATP. Vu is the slope of the linear least-squares regression (solid lines) and was 2.2±0.1 (SE) lengths · s-1 (r2=0.99) for ATP vs 3.3±0.4 lengths · s-1 (r2=0.95) for dATP.

In Vitro Motility
The greater increase in cardiac muscle V_u with dATP (compared with fast skeletal muscle V_u) could result simply from the increased F_max during contractions with dATP, especially considering the relatively large internal loads in cardiac muscle.³¹ Therefore, an independent measure of F-actin sliding speed was determined using an in vitro motility assay. This method provides a simple system to study crossbridge cycling using purified F-actin and HMM, eliminating the potential effects of internal loads that occur in trabeculae. Measurements of rhodamine phalloidin F-actin sliding speed (V_f) at saturating [NTP] (1 to 3 mmol/L) gave results quantitatively similar to those found in trabeculae and fast skeletal fibers (Table 2). V_f with cardiac HMM from untreated rats was >2-fold faster than V_f with cardiac HMM from PTU-treated rats. In both cases, dATP increased V_f by 70%, similar to the increase found for V_u. This compared with smaller, 30% increases by dATP in V_f with skeletal HMM and V_u in psoas fibers. When [NTP] was varied from 0.003 to 3 mmol/L, V_f for HMM from normal rats was increased at all [NTPs] >30 µmol/L, K_app was unchanged, and V_max was increased almost 2-fold by dATP (Figure 5). The similar K_app and much greater V_f indicate that dATP binds to cardiac myosin with affinity similar to that of ATP, but it enhances cardiac filament sliding speed.

View larger version (15K): [in this window] [in a new window]   Figure 5. In vitro motility assay of F-actin filament sliding speed (Vf) over HMM from untreated rat heart. Data are mean±SD of individual filament speeds (n ranged from 500 to 1500 filament paths), and solid lines are hyperbolic fits to the data (see Materials and Methods) used to estimate maximal Vf (Vf(*)) and Km. Data were fit (solid lines) with the equation y=Vf(*) [NTP]/([NTP]+Kapp). Vf(*) was 4.20±0.23 (±SE) vs 8.02±0.22 µm · s-1, and Kapp was 103±30 vs 109±9 µmol/L for ATP vs dATP, respectively.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The purpose of the present experiments was to determine whether the crossbridge transition rates controlling force and shortening in cardiac muscle are the same as in skeletal muscle. The major result from this study is that substitution of dATP for ATP as the contractile substrate reversibly increases maximal Ca²⁺-activated F_max, stiffness, k_tr, and V_u in both untreated and PTU-treated rat cardiac muscle (Table 2). Measures of crossbridge cycling with purified cardiac HMM, ie, both acto-HMM NTPase and V_f, were also increased by dATP (Tables 1 and 2). The rate of crossbridge cycling is also increased in rabbit psoas and soleus fibers, but without the increase in F_max seen in cardiac muscle (Table 2). The enhancement of F_max in cardiac muscle was not due to accumulation of dADP, because previous control measurements have shown that CK rephosphorylates dADP to dATP and ADP to ATP with a similar time course¹⁰ and, furthermore, k_tr, V_u, and V_f were faster. The results of this study demonstrate that dATP markedly enhances the mechanical properties of cardiac muscle and suggest that the process limiting F_max may differ in cardiac versus skeletal muscle.

PTU treatment decreased HMM NTPase, V_u, V_f, and k_tr in cardiac muscle (Tables 1 and 2), which is indicative of an MHC isoform shift from V1 to V3, without changes in other contractile protein isoforms, that has previously been shown by others.^{27 28 29 30} In support of a V1-to-V3 isoform shift, V_f using HMM from untreated versus PTU-treated rats in this study agrees well with V_f measured using HMM from T4 versus PTU-treated rabbits.³² Additionally, our measures of V_u and k_tr in cardiac and skeletal muscle agree with measurements made by others under similar conditions.^{33 34 35} Finally, the magnitude reduction of k_tr by PTU treatment in this study was similar to that for rat (primarily V1) versus guinea pig (primarily V3) cardiac trabeculae,³⁶ and k_tr was similar for PTU-treated cardiac muscle, rabbit soleus fibers (Table 2), and rat soleus fibers,³⁷ which also contain cardiac ß-MHC.³⁸ Our observation that chronic PTU treatment slows rat cardiac muscle contractile mechanics is consistent with previous documented shifts in cardiac MHC isoforms (from to ß). Importantly, the enhancement of contractile kinetics with dATP occurs regardless of myosin isoforms in both cardiac and skeletal muscle.

Mechanism of dATP Action
To identify the molecular basis for how dATP enhances the contractile kinetics of cardiac and skeletal muscle but enhances F_max only in cardiac muscle, we first examined recent structural and sequence information. The amino acid residues that form the ATP binding pocket are conserved in all of the MHC sequences relevant to this study.³⁹ The 2' oxygen of ADP in the x-ray crystal structure of scallop myosin complexed with MgADP makes contact with 2 residues, Tyr126 and Arg128.⁴⁰ The residues equivalent to Tyr126 and Arg128 in the scallop sequence are conserved in rabbit skeletal MHC (Tyr129 and Trp131), rat cardiac -MHC (Tyr127 and Trp129), and rat cardiac ß-MHC (Tyr128 and Trp130).³⁹ Thus, existing structural information and sequence conservation offer no explanation for the different effects of dATP on F_max in cardiac versus skeletal muscle, but are consistent with the enhancement of contractile kinetics in all of the preparations studied. This is evidenced by an enhancement of F_max by dATP in trabeculae from PTU-treated rat heart, but not in soleus fibers, whereas k_tr and V_u are enhanced in both preparations that contain ß-MHC (Table 2).

A more general approach to explore the effect of dATP on contractile performance is to fit the data to kinetic models of the crossbridge cycle. The dATP-induced increase in skeletal muscle crossbridge cycling rate, with no concomitant change in F_max, has been explained using the 2-state crossbridge model.¹⁶ Figure 6 shows the 2-state model in which the rate constants for crossbridge attachment and force generation (transition from weak to strong binding states) and crossbridge detachment are lumped into the apparent rates f_app and g_app, respectively. During maximal Ca²⁺ activation in fast psoas fibers, relatively small concomitant increases in both f_app and g_app could explain the lack of effect on F_max [f_app/(f_app+g_app)] and the slight increase in k_tr(f_app+g_app) with dATP.¹⁶ Our current results with soleus fibers can be explained by a similar mechanism. Because dATP increases F_max in cardiac muscle, in addition to increasing k_tr and V_u (Table 2), one possible explanation is that both f_app and g_app are increased (as in skeletal muscle), but f_app is increased more than g_app. Figure 6 predicts that a greater increase in f_app (relative to increased g_app) should result in an increased number of strongly attached, cycling crossbridges. This prediction is supported by our observed proportional increase in trabecular stiffness and F_max with dATP (see Results). A significant increase in crossbridge attachment and strong crossbridge binding with dATP, resulting from this kinetic mechanism, could increase the level of cardiac thin-filament activation, even at high levels of Ca²⁺.^{2 41}

View larger version (10K): [in this window] [in a new window]   Figure 6. Scheme of 2-state model in which the rate constants for crossbridge attachment and force generation (transition from weak to strong binding states) and crossbridge detachment are lumped into the apparent rates fapp and gapp, respectively.

Clinical Implications
The results of this study have potentially interesting implications for treating the force deficit associated with many cardiomyopathies. Our data thus far suggest that the enhancement of cardiac F_max makes dATP an attractive candidate for specific improvement of cardiac function. Thus, ribonucleotide reductase, the enzyme responsible for conversion of ATP to dATP,⁴² could be considered as a target for therapeutic treatment in heart failure, although the effects of upregulation of this enzyme on DNA synthesis and cellular metabolism need to be explored.

	Acknowledgments

 
This work was supported by National Institutes of Health Grants HL61683 and HL52558 and an American Heart Association Washington Affiliate Grant-in-Aid. We thank Dr Albert M. Gordon for reviewing the manuscript.

Received February 10, 2000; accepted April 28, 2000.

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