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(Circulation Research. 1996;78:893-902.)
© 1996 American Heart Association, Inc.

Articles

Decreased Energy Reserve in an Animal Model of Dilated Cardiomyopathy

Relationship to Contractile Performance

Ronglih Liao, Luigino Nascimben, Jan Friedrich, Judith K. Gwathmey, Joanne S. Ingwall

From the Cardiovascular Disease and Muscle Research Laboratories and the Department of Cellular and Molecular Physiology, Harvard Medical School (R.L., J.K.G.), the Charles A. Dana Research Institute and the Harvard-Thorndike Laboratory, Beth Israel Hospital (R.L., J.K.G.), and the NMR Laboratory for Physiological Chemistry, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School (L.N., J.F., J.S.I.), Boston, Mass.

Correspondence to Joanne S. Ingwall, PhD, NMR Laboratory for Physiological Chemistry, Department of Medicine, Brigham and Women's Hospital, Room BLI 247, 221 Longwood Ave, Boston, MA 02115. E-mail luigin@bustoff.bwh.harvard.edu.

	Abstract

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Abstract An animal model was used to test the hypothesis that in heart failure the decrease in the ability to resynthesize ATP through the creatine kinase (CK) reaction (which we call energy reserve) contributes to the inability of the heart to maintain its normal function and contractile reserve. One-week-old turkey poults were fed furazolidone for 14 days to induce dilated cardiomyopathy. Isolated Langendorff-perfused hearts from these myopathic animals showed a 73% decrease in baseline isovolumic contractile performance. Neither increasing [Ca²⁺]_o nor electrical pacing rate increased isovolumic contractile performance. Measured by ³¹P nuclear magnetic resonance magnetization transfer and chemical assay, ATP concentration was decreased by 23%, phosphocreatine concentration by 42%, CK enzyme activity by 34%, and the pseudo first-order rate constant for the CK reaction by 50%. Measured CK reaction velocity decreased by 71%. The reduced ability to increase cardiac performance in response to increasing [Ca²⁺]_o in hearts with lower CK reaction velocity was reproduced in part by feeding a separate group of turkey poults ß-guanidinopropionic acid to specifically reduce CK reaction velocity by decreasing guanidino substrate concentration. These hearts had normal baseline performance but blunted contractile reserve. These observations provide further support for the hypothesis that a decrease in energy reserve via the CK system contributes to reduced cardiac function in the failing heart.

Key Words: cardiomyopathy • cardiac performance • creatine kinase • phosphocreatine • ³¹P nuclear magnetic resonance magnetization transfer

	Introduction

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Creatine kinase (CK, EC 2.7.3.2) catalyzes the transfer of the phosphoryl group between PCr and ATP in striated muscle: PCr^2-+ADP^-+H⁺ ATP^2-+Cr. The rate of phosphoryl exchange between ATP and PCr in mammalian cardiac muscle is an order of magnitude faster than the rate of ATP resynthesis via oxidative phosphorylation¹ and many orders of magnitude faster than ATP resynthesis by de novo pathways.² The importance of the CK system in muscle energetics is clearly shown during the ATP supply-demand mismatch that occurs in hypoxia³ and ischemia,^{4 5} when phosphoryl transfer from PCr to ADP slows the rate of ATP depletion. The CK system also contributes to the ability of muscle to increase and sustain a higher level of work. During the last decade, experiments designed to specifically inhibit the CK reaction velocity in vivo using sulfhydryl inhibitors to reduce enzyme activity,^{6 7} guanidino substrate replacement,^{8 9 10 11} and gene ablation¹² have all demonstrated that decreasing CK reaction velocity limits the ability of striated muscle to increase its contractile performance.

The ability of striated muscle to increase contractile performance is described as contractile reserve. Because changes in work require concomitant changes in ATP synthesis rates, the biochemical correlate of contractile reserve is the ability to increase the rate of ATP synthesis in response to increased workload. We suggest that a useful term describing the biochemical analogue of contractile reserve is energy reserve. Because the rate of ATP resynthesis via the CK reaction is faster than oxidative phosphorylation, CK is the primary energy reserve system during high-workload conditions, when the heart uses its contractile reserve.

There are large changes in the CK system in failing cardiomyopathic hearts of humans and animals. Total CK activity (V_max) and total Cr and PCr contents are lower in failing than in nonfailing hearts.^{13 14 15 16 17 18 19 20 21} Since the velocity of the CK reaction is directly proportional to the product of CK activity (V_max) and [PCr], the decrease in total enzyme activity coupled with a decreased guanidino substrate content combine to decrease CK reaction velocity and hence the energy reserve of the heart.

The present study was undertaken to test the hypothesis that in heart failure the decrease in energy reserve through the CK system contributes to the inability of the heart to maintain its normal function as well as its contractile reserve. We tested this hypothesis in hearts of turkey poults with drug-induced dilated cardiomyopathy as a model of heart failure using two protocols. In the first protocol, using isolated Langendorff-perfused hearts, we directly measured CK reaction velocity in vivo using ³¹P-NMR magnetization transfer and simultaneously recorded isovolumic contractile performance. In the same hearts, we also measured tissue contents of Cr, ATP, and CK V_max using standard biochemical analyses. We also measured ATP synthesis rates estimated from oxygen consumption. Using this protocol, the relationship between cardiac function and CK reaction velocity was determined.

In a second protocol, to assess the specificity of changes in the CK system to explain these results, we decreased the content of the guanidino pool of normal myocardium by feeding the animals a Cr analogue, GPA. GPA, a competitive inhibitor of Cr transport,²² is a poor substrate for the CK reaction, and this reduces CK reaction velocity.^{9 10 11} This protocol allowed us to determine whether we could reproduce in whole or in part the changes in isovolumic contractile performance observed in the failing hearts by decreasing substrate concentration for the CK reaction. This approach allows us to test a possible mechanism for the decreased contractile performance characteristic of failing hearts.

	Materials and Methods

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Animal Protocols
Fz Treatment
One-day-old broad-breasted white turkey poults were obtained from Cuddy Farm, NC, and housed in temperature-controlled brooders. During the first 6 days, all birds were fed with normal turkey feed. On the seventh day, they were randomized into two groups. The control group was maintained on a normal ration free from any additives. The second group (Fz-DCM group) was maintained on a similar ration, but it also contained Fz at a concentration of 700 ppm. Fz has been shown to reliably produce a dilated cardiomyopathy in turkey poults.^{23 24}

Echocardiograms were performed to confirm the presence of in vivo cardiac dilatation and decreased ejection fraction in Fz-treated animals.²⁵ By 3 weeks of age, cardiac dilatation was observed in all Fz-DCM poults but in none of the control poults. At this time, the turkey poults were killed. Body weights were 398±12 g for the control group and 289±12 g for the Fz-DCM group (P<.0001). Hearts of Fz-DCM animals exhibited no observable fibrotic areas, but their left ventricles were grossly dilated.

ß-GPA Treatment
Another cohort of 1-day-old turkey poults was randomly distributed into two groups. One group was maintained as a control group using the protocol described above. The second group was treated with GPA for 20 days, starting at 2 days of age. The analogue was administered by mixing GPA with normal turkey feed (0.5% [wt/wt]) and adding GPA 200 mg/kg body wt orally each day. These doses are similar to those used in previous studies that produced significant decreases in Cr and PCr in rat hearts.^{9 10 11} Turkey poults were allowed feed and water ad libitum. The mean body weight of GPA-fed poults was 422±18 g (P=.32 versus control poults). Hearts of GPA-treated poults exhibited no observable morphological abnormalities and were neither hypertrophied nor grossly dilated.

All animals were cared for according to the guidelines of the American Physiological Society.

Turkey Heart Preparation
Turkey poults were anesthetized intravenously with sodium pentobarbital (65 mg/kg body wt) at 19 to 21 days of age. Intravenous heparin (100 U/kg body wt) was also administered before quickly excising the heart and placing it in ice-cold buffer. The heart was then attached to a perfusion apparatus, and retrograde perfusion was begun through the aorta (the Langendorff mode) at 41°C (the normal body temperature of the turkey) and a constant hydrostatic pressure of 85 mm Hg. Hearts perfused at this pressure were able to maintain a maximal ratio of [PCr] to [ATP] for at least 120 minutes. The perfusate was phosphate-free Krebs-Henseleit buffer containing (mmol/L) NaCl 113, KCl 4.7, CaCl₂ 2.5 or 5.5, MgSO₄ 1.2, EDTA 0.5, NaHCO₃ 25, sodium pyruvate 5, and glucose 11, along with 6.25 g/L dextran. The perfusate was saturated with a gas mixture containing 95% O₂/5% CO₂ to maintain a pH of 7.4. Coronary flow was measured by collecting timed coronary sinus effluent samples in a calibrated cylinder.

Measurement of Isovolumic Contractile Performance
Left ventricular pressure and heart rate were continuously recorded by connecting a water-filled latex balloon placed in the left ventricle to a Statham DB23 pressure transducer. The balloon was inflated to set left ventricular end-diastolic pressure at 10±2 mm Hg for all hearts. Left ventricular-developed pressure (the difference between systolic and diastolic pressures) was used as an indicator of the contractile state of the heart. Previous studies^{25 26} have shown that for the failing Fz-DCM hearts, systolic and developed pressures are relatively insensitive to changes in end-diastolic pressure produced by varying the balloon volume. For the present study, we chose to use similar loading conditions (end-diastolic pressure). This was obtained using a balloon that was four times larger for the Fz-DCM hearts. In some of the experiments, the hearts were electrically paced. Isovolumic contractile performance was estimated as the product of heart rate and developed pressure (RPP, in millimeters of mercury per minute).

In order to better estimate cardiac work in severely dilated Fz-DCM hearts, we also adjusted RPP for the geometric parameters of the left ventricle according to the Laplace equation. The Laplace equation is as follows: wall stress=P·r/(2d), where P is systolic pressure, r is the radius of the short axis of the left ventricular cavity, and d is the thickness of the left ventricular wall. For the calculation made here, we used RPPs measured in the present study for control and Fz-DCM hearts and the average geometric constants (r=2.2 mm and d=4.5 mm for control hearts, and r=5.5 mm and d=2.8 for Fz-DCM hearts), which were previously measured using an echocardiographic method in hearts maintained under identical conditions.²⁷

Measurement of Isovolumic Contractile Performance and Oxygen Consumption With High Ca²⁺ Challenge
Ten turkey poults (5 control turkey poults and 5 Fz-DCM turkey poults) were used for the measurements of oxygen consumption. An oxygen meter (ORION model 860) was used to determine oxygen concentration in the perfusate. Oxygen consumption in perfused hearts was monitored by sampling the oxygen concentration of aortic inflow and pulmonary vein outflow perfusates. Cardiac performance (RPP) and oxygen consumption for each heart were determined at baseline ([Ca²⁺]_o of 2.5 mmol/L supplied for 20 minutes) and then at an increased [Ca²⁺]_o of 5.5 mmol/L (perfused for 15 minutes). [Ca²⁺]_o was then returned to baseline (2.5 mmol/L) for 15 minutes before challenging each heart by electrically pacing at 300 bpm. MVO₂ values of perfused hearts were calculated as MVO₂=(O₂·CF)/dry weight of heart, where O₂ is the difference in oxygen concentrations between aortic inflow and pulmonary vein outflow perfusate and CF is coronary flow.²⁸ The measured value of 0.218 was used for the ratio of dry weight to wet weight of failing and nonfailing turkey hearts. Oxygen consumption is expressed in micromoles of O₂ per minute per gram of dry weight. When converting to ATP synthesis rates, we assumed that 6 moles of ATP were produced for every mole of O₂ consumed.

Isovolumic contractile performance without oxygen consumption was measured in a second cohort of 10 (5 control and 5 GPA-treated) turkey poults at baseline extracellular Ca²⁺ of 2.5 mmol/L and after 15 minutes of challenge with high extracellular Ca²⁺ of 5.5 mmol/L.

³¹P-NMR Spectroscopy
³¹P-NMR experiments were carried out on 7 control and 4 Fz-DCM hearts. Each isolated perfused nonpaced heart was placed in a 30-mm NMR probe and inserted into the bore of an 8.4-T magnet (Oxford Instruments). The magnet was connected to an NT360 Spectrometer interfaced with a 1280 computer (Nicolet Instruments) operating in pulsed Fourier transform mode. An 18-channel Oxford Instrumentation shim supply was used to homogenize the magnetic field by minimizing the width of the ¹H signal. ³¹P-NMR spectra were obtained at 145.75 MHz. Magnetization transfer was performed by applying a low-power (B₁ field of 30 Hz) radiofrequency pulse centered at the [-P]ATP resonance for progressively longer time periods, from 0 to 4.8 seconds. The low-power pulse was calibrated by irradiating the [-P]ATP peak to ensure complete saturation, followed by irradiation downfield at a frequency equidistant from the PCr resonance to ensure that there was negligible off-resonance saturation of the PCr resonance. Magnetization transfer spectra were obtained by signal averaging 64 scans of high-power broadband 58° (70-microsecond) read pulses after the low-power narrow-band saturation pulse was interleaved in groups of eight and separated by a constant delay of 7 seconds, including the saturation pulse time. This increased the probability that effects due to any changes in contractile function or rhythm during the signal averaging were equally distributed among the spectra. A complete saturation transfer experiment was acquired in 45 minutes. Control spectra without presaturation, obtained over 4.5 minutes by signal averaging 104 scans of 58° read pulses separated by an interscan delay of 2.6 seconds, were acquired immediately before and after the magnetization transfer experiment to assess the stability of the preparation over the course of the experiment. A spectral width of 6000 Hz was used. Individual free induction decays were zero-filled from 1024 to 2048 data points²⁹ and weighted with a 20-Hz line-broadening decaying exponential before Fourier transformation.

Biochemical Measurements
At the end of the NMR experiment, each heart was freeze-clamped and stored at -70°C for subsequent biochemical assays. Frozen tissue was homogenized in a phosphate buffer containing 1 mmol/L EDTA and 1 mmol/L ß-mercaptoethanol at pH 7.4. Aliquots were removed for measurement of protein content according to the method of Lowry et al,³⁰ with bovine serum albumin used as the standard, and for measurement of total Cr content, a fluorometric assay was used according to the method of Kammermeier.³¹ Triton X-100 was then added to the homogenate at a final concentration of 0.1% for analysis of CK activity. Total CK activity (V_max) was measured with the coupled enzyme scheme of Rosalki³² at 30°C using a Calbiochem-Behring CK-NAC SVR kit. The ratio of activities at 41°C to 30°C was empirically determined to be 2.18. A separate portion of the freeze-clamped heart was used to determine [ATP] by HPLC.³³

Data Analysis
Metabolite Concentrations
Integrated signal intensities in ³¹P-NMR spectra corresponding to amounts of ATP, PCr, and P_i in each heart were measured using the NMR1 curve-fitting routine (New Methods Research Inc).

[ATP] from HPLC is expressed as nanomoles per milligram Lowry protein. Lowry protein minimizes the contribution from proteins found in the extracellular space, because these proteins contain relatively low amounts of aromatic amino acids, the target for this assay. It thus approximates the myocyte protein content. Lowry protein content differed between the control group (0.125±0.004 mg protein per milligram wet weight) and the Fz-DCM group (0.087±0.003 mg protein per milligram wet weight) (P<.0001). For the control group, [ATP] determined by HPLC (nanomoles per milligram protein) was converted to cytosolic concentration (millimolar) using myocyte protein content and the literature value of 0.5 mL intracellular water per gram wet weight obtained for isolated perfused hearts of small animals.^{34 35} The same conversion was carried out for the Fz-DCM hearts, but wet weight was scaled by the control Lowry protein content. This approach ensures that calculation of substrate and protein concentrations take into account any replacement of myocyte volume by fibrotic tissue in the failing hearts. We made the conservative assumptions that the Fz-DCM myocytes also had an intracellular water concentration of 0.5 mL/g wet wt and that the hypertrophy that occurs in these myocytes²⁴ does not alter the fractional cell volume of intracellular water.³⁶ These assumptions and the scaling procedure minimize any decreases in tissue metabolite contents by providing an estimate of the maximum intracellular concentrations in the Fz-DCM hearts. These assumptions were also used when converting MVO₂ (micromoles oxygen consumed per minute per gram dry weight) to ATP synthesis rates (millimoles per liter per second).

[PCr] was determined by multiplying [ATP] by the ratio of PCr to [ß-P]ATP determined from the NMR spectra after correction for differential incomplete relaxation based on measured relaxation times for ATP and PCr.³⁷ pH_i of each perfused heart was measured by comparing the chemical shift between the P_i and PCr resonances with values obtained from a standard curve mimicking the intracellular ionic strength and pH.

Cytosolic free [ADP] was estimated by using [ATP], [PCr], and [H⁺] measured in the intact beating heart by ³¹P-NMR spectroscopy and the total Cr measured chemically in heart homogenates and by assuming an equilibrium constant (1.66x10⁹·[H⁺])³⁸ for the CK reaction:

Measured CK Reaction Velocity
The velocity of the forward CK reaction (ie, in the direction of ATP synthesis), V_for, equals k_for[PCr]. The magnetization transfer measurements of the forward CK reaction were analyzed according to the two-site chemical exchange model,³⁹ providing a measurement of k_for. The two-site chemical exchange model is applicable because PCr participates in no reaction other than that catalyzed by CK. The rate and extent of decay of the PCr signal is a balance between two opposing mechanisms: (1) the rate of chemical exchange determined by k_for, which acts to promote the signal decay, and (2) the rate of relaxation of exchanged saturated signal determined by T₁, the intrinsic longitudinal relaxation time constant for PCr, which opposes the signal decay. At short saturation times, the signal decay rate is greater, resulting in an exponential decay to a new steady state magnetization at longer saturation times, when the two opposing transfer rates become equal. Variance-weighted nonlinear regression was used to fit the integrated signal intensity of PCr magnetization, M_t, which decays from M₀ to M, and the time of saturation at [-P]ATP, t, to a three-parameter single exponential as follows:

Values of k_for and T₁ were calculated by solving two simultaneous equations:

and

Multiplying k_for by [PCr] yields the measured reaction velocity:

Statistical Analysis
Variance-weighted nonlinear regression was used to fit the observed decay data in the PCr magnetization signal to a three-parameter decaying exponential. Student's t test was used to compare between two groups of data. A value of P<.05 was taken as statistically significant. All data are presented as mean±SEM.

	Results

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Isovolumic Contractile Performance in Fz-DCM and Control Hearts
Baseline Cardiac Performance
Isovolumic contractile performance in unpaced hearts was estimated as RPP (Table 1). Isovolumic contractile performance of the Fz-DCM hearts was 73% lower than in the control hearts. This is primarily due to lower left ventricular developed pressures in the Fz-DCM hearts (72% lower). However, RPP values that have been adjusted for changes in chamber radius and wall thickness for control hearts (6200±600 mm Hg/min) and Fz-DCM hearts (7000±1900 mm Hg/min) are similar (P=.64). There was no difference in either the coronary flow or heart rate between control and Fz-DCM hearts.

View this table: [in this window] [in a new window]   Table 1. Baseline Cardiac Performance of Fz-DCM and GPA-Treated Hearts Compared With Control Hearts

Isovolumic Contractile Performance and MVO₂ at Baseline and With High Ca²⁺ Challenge and Pacing
Baseline MVO₂ rates for control and Fz-DCM hearts were similar, correlating with comparable values for RPP normalized for changes in geometry in control and Fz-DCM hearts (Table 2). The baseline ATP synthesis rate was 41% higher in the Fz-DCM hearts.

View this table: [in this window] [in a new window]   Table 2. Cardiac Performance, Oxygen Consumption, and ATP Synthesis With Increased [Ca2+]o and Pacing of Control and Fz-DCM Hearts

When control hearts were challenged with high [Ca²⁺], MVO₂ and isovolumic contractile performance increased by 64% and 38%, respectively. In contrast, when Fz-DCM hearts were challenged with high [Ca²⁺], MVO₂ increased (+31%), but isovolumic contractile performance remained unchanged. Similarly, pacing Fz-DCM hearts did not increase isovolumic contractile performance.

The CK System in Fz-DCM and Control Hearts
CK Activity and Substrate Concentrations
Representative ³¹P-NMR spectra obtained from control and Fz-DCM hearts are shown in Fig 1. These spectra show that the total amounts of both ATP and PCr and their ratio are lower in the Fz-DCM heart when compared with the control heart. This is the case in spite of the increase in mass of the failing heart^{25 26 27} and shows that both [ATP] and [PCr] are lower in the Fz-DCM heart. The integrated area for P_i was small for both the failing and nonfailing hearts.

View larger version (24K): [in this window] [in a new window]   Figure 1. Representative 31P-NMR spectra of control (CON, A), Fz-DCM (B), and GPA-treated (C) hearts. The major resonances are assigned as monophosphate ester (MPE), Pi, PCr, -, -, and ß-phosphates of ATP, and NAD. Integrated peak intensities are proportional to metabolite content. The Fz-DCM spectrum illustrates that the PCr-to-ATP ratio and the PCr and ATP contents in the Fz-DCM heart are lower than in the nonfailing CON heart. In the spectrum of the GPA heart, the phosphorylated GPA resonance signal is located 0.5 ppm upfield from the PCr signal. When compared with the CON spectrum, this spectrum illustrates a significant replacement of PCr by phosphorylated GPA (P-GPA), a poor substrate for the CK reaction (PCr was 50% lower on average), but an unchanged ATP content.

Total CK activity (V_max) and the cytosolic concentrations of the substrates and products of the CK reaction for all control and Fz-DCM hearts determined using ³¹P NMR and biochemical methods are shown in Table 3. Total CK activity (V_max) in Fz-DCM hearts was 30% lower than in control hearts (P=.006). [PCr], [Cr], and [ATP] were all lower in Fz-DCM hearts than in control hearts. In addition, the ratio of [PCr] to [ATP] in Fz-DCM hearts (0.94±0.05) was lower than that in control hearts (1.25±0.04) (P=.0006). Estimates for [ADP] from the CK equilibrium expression were the same for the two groups. pH_i was 7.11±0.04 for both groups.

View this table: [in this window] [in a new window]   Table 3. The CK System: Enzyme Activity, Substrate Concentrations, and Flux Through the CK Reaction in Control and Fz-DCM Hearts

It is important to emphasize that the concentrations shown in Table 3 for failing hearts are likely to be maximum values and that any differences between control and Fz-DCM hearts are minimized by expressing values as cytosolic concentrations. By scaling metabolite concentrations by the decrease in Lowry protein content, we take into account the decreased content of myocyte protein (presumably due to increased fibrosis) in the failing hearts, thereby reducing any apparent differences in metabolite content. For example, based on the NMR spectra such as shown in Fig 1, the average amount of ATP in the Fz-DCM hearts was 47% lower than in the control hearts. By taking the 27% decrease in protein content (0.087 versus 0.125 mg protein per milligram wet weight) into account, the decrease in cytosolic [ATP] is 23% lower (6.5 versus 8.5 mmol/L).

Experimentally Determined V_for
Representative stacked plots for ³¹P magnetization transfer experiments for Fz-DCM and control hearts are shown in Fig 2, and values characterizing V_for in vivo for all hearts studied are shown in Table 3. The value for k_for derived from the exponential decay of PCr magnetization was 50% lower for Fz-DCM hearts compared with control hearts (P=.0003). V_for (k_for[PCr]) was 71% lower (P<.0001).

View larger version (34K): [in this window] [in a new window]   Figure 2. Representative stacked plots of 31P-NMR spectra of control (CON) and Fz-DCM hearts. The major resonances are assigned as in Fig 1. These plots illustrate the magnetization transfer method used to measure kfor for the CK reaction in the direction of ATP synthesis. The saturation pulse was applied at the resonance frequency for [-P]ATP. The saturation duration is shown on the right of each spectrum. The spectrum with no [-P]ATP saturation is shown as the first and last spectrum in each stacked plot. Values for T1, the spin-lattice relaxation constant, for PCr were the same in CON and Fz-DCM hearts (data not shown). Thus, the differences in the decay rates of the PCr signal reflect differences in kfor of the CK reaction in Fz-DCM hearts.

Relationship Between V_for and Cardiac Performance
The magnitude of the decrease in V_for in Fz-DCM hearts is similar to the decrease observed for isovolumic contractile performance (RPP), suggesting that the impaired CK system may contribute to the decreased cardiac performance in the Fz-DCM hearts. To better characterize this relationship, we analyzed the relationship between V_for and isovolumic contractile performance (RPP) with and without adjustment for geometry for both failing and nonfailing hearts.

Fig 3A plots V_for against isovolumic contractile performance measured as RPP for all hearts studied. Reaction velocity and isovolumic contractile performance are both greater in the control hearts compared with the Fz-DCM hearts. Fig 3B plots V_for against RPP adjusted for differences in wall stress of each heart. This plot shows that V_for of the Fz-DCM hearts is decreased even though the left ventricles of the two groups are experiencing similar wall stresses.

View larger version (10K): [in this window] [in a new window]   Figure 3. A, Flux through the CK reaction plotted against cardiac performance (RPP) for control (CON, ) and Fz-DCM () hearts, illustrating that both flux and RPP are decreased in the Fz-DCM hearts. B, Flux through the CK reaction plotted against cardiac performance corrected for geometric wall stress parameters, ie, RPPx(r/2d), illustrating that the left ventricles of the Fz-DCM hearts exhibit decreased flux despite experiencing comparable wall stresses.

These results suggest that one or more of the biochemical determinants of CK reaction velocity (total CK activity, purine, and/or guanidino substrate contents) in the Fz-DCM hearts may contribute to impaired baseline cardiac performance. Impaired CK reaction velocity may also contribute to the decreased cardiac reserve observed in these failing hearts. These possibilities can be tested, at least in part, by specifically decreasing the guanidino substrate pool size and measuring the effects on isovolumic contractile performance.

Effect of GPA
Fig 1C shows a representative spectrum of an isolated perfused heart obtained from a GPA-treated animal. The spectrum of the GPA-treated heart shows a phosphorylated GPA resonance peak 0.5 ppm upfield from the PCr resonance. The spectrum also shows that the PCr resonance area is markedly reduced. The average decrease in PCr measured for animals treated for 3 weeks in this study is 50%. [ATP] was unchanged in the GPA-treated hearts compared with the control hearts, similar to that observed by others in rodent hearts with even greater PCr replacement.^{9 10 11}

Fig 4A shows baseline RPP and RPP with high Ca²⁺ challenge for control, Fz-DCM, and GPA-treated hearts. Fig 4B shows isovolumic contractile performance changes as the percentage increase from baseline, illustrating that the percentage increase in RPP of GPA-treated hearts is between that for control and Fz-DCM hearts. GPA-treated hearts had indistinguishable baseline cardiac contractile performance compared with control hearts (Table 1). When the GPA-treated hearts were challenged with high Ca²⁺, they showed some increase in isovolumic performance (P=.0026 versus baseline GPA), but they were unable to increase their cardiac performance to the same level as control hearts analyzed at the same time (P=.015 versus high Ca²⁺ control).

View larger version (14K): [in this window] [in a new window]   Figure 4. A, Absolute RPP at baseline ([Ca2+]=2.5 mmol/L) and with inotropic challenge ([Ca2+]=5.5 mmol/L) for control (CON, ), GPA-treated (), and Fz-DCM () hearts. B, Relative change in cardiac performance (RPP) from baseline with inotropic challenge. The GPA-treated hearts exhibit an intermediate increase in cardiac performance between that of the failing Fz-treated hearts and the control hearts.

Thus, decreasing the guanidino substrate concentration of the CK reaction without changing enzyme activity by supplying GPA to normal animals did not affect baseline function. However, this approach partially reproduced one component of the decreased contractile performance observed in failing Fz-DCM hearts, namely, impaired ability to increase RPP in response to increased stress.

	Discussion

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In the present study, we determined isovolumic contractile performance at baseline and in response to inotropic challenge in an animal model of cardiomyopathy and related changes in contractile performance to changes in high-energy phosphate content and synthesis rates. The failing hearts exhibited decreased baseline performance compared with hearts isolated from age-matched control animals and perfused under the same conditions. Furthermore, unlike the control hearts, they were unable to increase their contractile performance either when paced at higher heart rates or when inotropically challenged with high extracellular Ca²⁺. Impaired baseline contractile performance and the inability to recruit contractile reserve were associated with decreases in ATP and PCr contents and in the rate of phosphoryl exchange between ATP and PCr catalyzed by CK but not with ATP synthesis via oxidative phosphorylation. Partially replacing the myocardial PCr pool with a poorly hydrolyzable guanidino analogue (GPA) in control animals reproduced some of the characteristics of the failing hearts, namely, decreased PCr contents and their inability to respond normally to inotropic challenge elicited by high [Ca²⁺]_o. It did not reproduce the decrease in baseline cardiac performance observed in Fz-DCM hearts. These results provide further support for the hypothesis that there is a relationship between decreased energy reserve and decreased contractile reserve in the failing heart.

Isovolumic Contractile Performance, Wall Stress, and MVO₂ in Isolated Failing Fz-DCM Hearts
Consistent with decreased cardiac performance of failing myocardium in humans and in mammalian animal models of heart failure, baseline isovolumic contractile performance measured as RPP was 73% lower in the Fz-DCM turkey hearts. Decreased performance was primarily due to a decrease in developed pressure. In spite of this large decrease in developed pressure, RPP adjusted for geometric parameters using the Laplace relation was not different in the Fz-DCM hearts. It seems likely that hypertrophy and dilatation of the Fz-DCM hearts combine to maintain normal wall stress in these hearts. Coronary flow and MVO₂, also measured at baseline, were similar for control and Fz-DCM hearts. Thus, oxygen supply and oxygen utilization correlate better with wall stress than with RPP. These observations are consistent with work by others showing that MVO₂ is more closely correlated with pressure-volume area than with RPP.^{40 41}

With inotropic challenge, both isovolumic contractile performance and MVO₂ increased in the control hearts. In contrast, in the Fz-DCM hearts, isovolumic contractile performance remained unchanged while MVO₂ increased. Thus, these failing hearts were unable to recruit their contractile reserve. Recent results using a genetic model of heart failure, namely, the cardiomyopathic hamster, have shown a similar abnormal response of the heart to inotropic challenge.⁴²

ATP Content and Synthesis Rates From the Major Pathways for ATP Synthesis
Using ³¹P-NMR spectroscopy combined with classic tools of biochemistry and physiology, we measured ATP content and oxygen-dependent and oxygen-independent ATP synthesis rates in control and failing hearts. Both ³¹P-NMR spectroscopy, which measures the amount of ATP in the whole heart, and HPLC measurement, which measures ATP in biopsy specimens obtained from ventricular tissue, show that ATP content in Fz-DCM hearts is lower (23%) than in aged-matched control hearts. This is the case even when normalized to account for any differences in extracellular water and protein contents. Because of problems quantifying absolute concentrations of metabolites using ³¹P-NMR spectra obtained from the human heart, it is not yet clear whether [ATP] is lower in failing human myocardium. Results using ³¹P-NMR spectroscopy to study animal models of heart failure are therefore particularly useful.

The results presented here for drug-induced heart failure in the turkey poult showing a 23% decrease in ATP content are in good agreement with results obtained for a genetic model of heart failure in a small mammal. Measured both by ³¹P NMR and HPLC, ATP content was 28% lower in the 10-month-old failing hamster heart.¹³ In contrast, ³¹P-NMR studies of two other small animal models of heart failure, namely, the 18-month-old spontaneously hypertensive rat⁴³ and the remodeled rat heart 12 weeks after infarction, did not detect decreases in ATP either regionally⁴⁴ or globally.²¹ In these cases, however, the NMR measurements were made before there was evidence of severe failure in these animals. Taken together, these results suggest that decreases in [ATP] may not occur until the heart is in severe failure.

Because of the importance of maintaining normal ATP levels to support contraction and excitation, there are many pathways for ATP synthesis in the heart. The major oxygen-dependent pathway is oxidative phosphorylation. In the present study, we estimate the rate of ATP synthesis from oxidative phosphorylation from MVO₂ measurements. Comparison of ATP synthesis rates measured as MVO₂ and by direct phosphoryl transfer between P_i and ATP by ³¹P-NMR spectroscopy have shown good agreement for control hearts.¹ Under all conditions studied (at baseline, with increased heart rate, and with increased inotropic stress due to increased [Ca²⁺]_o), ATP synthesis rates estimated in this way were higher in Fz-DCM than in control hearts. A surprising result of the present study is that even an apparent increased rate of oxygen-dependent ATP production failed to maintain normal ATP levels in the failing heart.

We did not measure ATP production from substrate level phosphorylation via glycolysis, but we found no differences in the tissue contents of the major glycolytic proteins phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, and lactate dehydrogenase (data not shown). Mirsalimi et al⁴⁵ found decreased phosphofructokinase and lactate dehydrogenase activities in 6-week-old turkey hearts treated with Fz for 4 weeks. The apparent discrepancy may be explained by differences in normalization (myocardial mass rather than protein content), maturation, or duration of Fz treatment. Our results suggest that the capacity for ATP production via glycolysis is unchanged in the failing heart. However, since the rate of glycolysis and V_max of the glycolytic proteins do not always correlate,⁴⁶ glycolytic rates may differ in failing and nonfailing myocardium.

A major observation of the present study is that both the capacity for ATP synthesis via the CK system (V_max) and the unidirectional V_for directly measured using ³¹P-NMR magnetization transfer are lower in the Fz-DCM heart. The decrease in V_max for the CK reaction is not due to a nonspecific decrease in the content of all proteins, since neither the tissue activities of glycolytic enzymes (see above) nor the mitochondrial enzyme (citrate synthase) differ in control or Fz-DCM hearts (data not shown). In the control hearts, V_for is approximately an order of magnitude greater than the rate of ATP synthesis measured as MVO₂ (7.8 versus 0.9 mmol/L per second), similar to what has been measured in isolated perfused mammalian hearts.^{1 44} In contrast, in the Fz-DCM hearts, V_for is only slightly greater than the rate of ATP synthesis (2.3 versus 1.3 mmol/L per second). Thus, not only is the ATP content lower in the failing heart, but the ability to rapidly resynthesize ATP via the CK reaction is also impaired.

CK System in Failing Fz-DCM Hearts
Results from both the noninvasive tool of ³¹P-NMR spectroscopy and chemical assay show that the Fz-DCM hearts have decreased energy reserve via the CK reaction. This is shown by a decrease in the capacity for ATP synthesis via the CK reaction, measured as tissue enzyme activity (V_max, 34% lower) and by a decrease in tissue content of PCr (42% lower). Since flux through the CK reaction is proportional to the product of V_max and PCr, these results predict that V_for is reduced by as much as 60%. Direct measurement of V_for in the intact beating heart confirms this prediction. k_for, measured using magnetization transfer, was decreased by 50% in failing versus nonfailing hearts. This results in a phosphoryl transfer rate that is 71% lower. The values of phosphoryl transfer of 60%, estimated by the product of V_max and [PCr], and 71% obtained by direct measurement are in good agreement.

Large decreases in CK activity and Cr content have been measured in biopsy samples obtained from patients with dilated cardiomyopathy.^{15 47 31}P-NMR spectroscopy measurements have shown a decreased ratio of PCr to ATP^{18 19 48} in the failing human heart. Given our results showing that ATP content is also lower in failing myocardium, the decreases in PCr are likely to be even greater than indicated by the PCr-to-ATP ratio. In addition to the decreased CK activity and Cr content, decreased phosphoryl transfer has been measured in myopathic hamster hearts¹³ and rat hearts 12 weeks after coronary artery occlusion.²¹ These results, combined with our findings for failing turkey hearts, show that the defects in the CK system occur independently of species, maturation, or pathogenesis and thus are characteristic of failing myocardium.

To study the contribution of decreased energy reserve via the CK reaction on impaired cardiac performance in the Fz-induced dilated cardiomyopathic turkey hearts in another way, we perturbed the CK system in a group of control animals by chronically replacing the Cr pool with GPA. Others have shown that partially replacing the Cr pool in this way decreases V_for.^{10 11} Although we were unable to quantify V_for in GPA-supplied turkey hearts because of the substantial overlap of the PCr and phosphorylated GPA resonances, we have confirmed these observations qualitatively. The 50% decrease in PCr observed in the GPA-treated hearts studied here was slightly greater than the 42% decrease in PCr in the Fz-DCM hearts. However, unlike the Fz-DCM hearts, in which enzyme activity was also decreased (V_max was decreased by 34%), GPA does not affect CK activity.⁸ Thus, since GPA treatment reproduced only the decrease in substrate content without affecting enzyme activity, GPA treatment produced less of a perturbation of the CK system than we observed with Fz-DCM. In addition, in the GPA-treated hearts, ATP levels were unchanged. If decreasing the velocity of the creatine kinase reaction, either directly or indirectly, does make a major contribution to the contractile reserve of the heart, we would expect the ability to recruit contractile reserve of GPA-treated hearts to be between those of control and Fz-DCM hearts. This is what was observed. Baseline cardiac performance in the GPA-treated hearts was unchanged compared with control hearts, and GPA-treated hearts showed some ability to increase performance with increased extracellular Ca²⁺. However, this increase was lower than the increase in function measured for the control hearts. Thus, partial chronic replacement of the Cr pool blunts the contractile reserve of the heart.

These results are in agreement with most of the results obtained by others in rats treated with GPA for longer time periods (6 to 10 weeks), resulting in greater replacement of PCr by GPA (80% to 90%): Shoubridge et al⁹ showed unchanged cardiac performance at low workloads, whereas Kapelko et al¹⁰ showed decreased performance at higher workloads. However, in conflict with these observations, Zweier et al¹¹ showed decreased performance at all workloads. The basis for the apparent discrepancy is unknown but could be due to important differences in choice of analogue, dose, and duration of supply.

The relationship between energy reserve via the CK reaction and contractile reserve has been previously studied in normal mammalian hearts using three strategies: (1) Acute specific chemical inhibition of CK activity in intact rat hearts with iodoacetamide^{7 37 41} resulted in decreased V_max and the loss of contractile reserve. (2) Chronic replacement of Cr with poorly hydrolyzable Cr analogues also reduced V_for and blunted contractile reserve of the heart.^{10 11} (3) Permanent reduction in CK activity by deleting the gene for M (muscle)-CK monomer blunted the ability of skeletal muscle to sustain acute or burst work.¹² In the present study, we show that in the failing heart both CK activity and substrate pool are chronically decreased, thereby decreasing CK reaction velocity. Contractile reserve is also decreased. Partially decreasing the guanidino substrate content in normal turkey hearts also decreased contractile reserve of the heart. The link between decreased CK reaction velocity and decreased contractile reserve may be direct (rapid resynthesis of ATP needed for contraction) or indirect (via unknown mechanisms) and merit future studies. Whichever the case, the results presented here provide further evidence suggesting a relationship between decreased energy reserve via the CK system and decreased contractile reserve.

	Selected Abbreviations and Acronyms

 

CK = creatine kinase Cr = creatine DCM (in association = dilated cardiomyopathy with treatment group) Fz = furazolidone GPA = ß-guanidinopropionic acid kfor = pseudo first-order forward rate constant M0, M = magnetization at zero and infinite saturation time Mt = magnetization at saturation time t MVO2 = rate of oxygen consumption NMR = nuclear magnetic resonance PCr = phosphocreatine RPP = rate-pressure product T1 = relaxation time constant Vfor = velocity of the forward CK reaction

	Acknowledgments

 
This study was supported in part by National Institutes of Health grant R01 HL-49574 (Dr Gwathmey). Dr Gwathmey was an Established Investigator of the American Heart Association. Dr Liao was supported by National Institutes of Health training grant 5T32 HL-07374-15. Dr Nascimben was supported by a Merck Sharp & Dohme Italia S.p.a. fellowship award. Dr Friedrich was supported by Harvard-MIT Division of Health Science and Technology, Johnson & Johnson, and Research Fund fellowships. We thank Dr Rong Tian for assistance with the interpretation of the oxygen consumption results and Dr Laura Stewart for helping to set up the Langendorff preparation for the turkey hearts. We also thank Jonathan Rose, Ilana Reis, Leslye Howell, and Kim H. Ky for technical assistance, Cuddy Farm for their support, and Drs Carl S. Apstein and Pierre D. Dos Santos for helpful comments.

	Footnotes

 
This manuscript was sent to Harold C. Strauss, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received October 11, 1995; accepted February 16, 1996.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Bittl JA, Ingwall JS. Reaction rates of CK and ATP synthesis in the isolated rat heart. J Biol Chem. 1985;260:3512-3517. [Abstract/Free Full Text]

2. Zimmer H-G, Trendelenburg C, Kammermeier H, Gerlach E. De novo synthesis of myocardial adenine nucleotides in the isolated perfused rat heart. Circ Res. 1973;32:635-642. [Abstract/Free Full Text]

3. Bittl JA, Balschi JA, Ingwall JS. Contractile failure and high-energy phosphate turnover during hypoxia: ³¹P-NMR surface coil studies in living rat. Circ Res. 1987;60:871-878. [Abstract/Free Full Text]

4. Neubauer S, Hamman BL, Perry SB, Bittl JA, Ingwall JS. Velocity of the CK reaction decreases in postischemic myocardium: a ³¹P-NMR magnetization transfer study of the isolated ferret heart. Circ Res. 1988;63:1-15. [Abstract/Free Full Text]

5. Cave AC, Eberli FE, Ngoy S, Rose J, Ingwall JS, Apstein CS. Increased glycolytic substrate protects against ischemic diastolic dysfunction: ³¹P^-NMR studies in the isolated blood perfusion rat heart. Circulation. 1993;88(suppl I):I-43. Abstract.

6. Fossel ET, Hoefeler H. Complete inhibition of creatine kinase in isolated perfused rat hearts. Am J Physiol. 1987;252(Endocrinol Metab 15):E124-E130.

7. Hamman BL, Bittl JA, Jacobus WE, Allen PD, Spencer RS, Tian R, Ingwall JS. Inhibition of the creatine kinase reaction decreases the contractile reserve of the isolated rat heart. Am J Physiol. 1996;269(Heart Circ Physiol 38):H1030-H1036.

8. Chevli R, Fitch CD. ß-Guanidinopropionate and phosphorylated ß-guanidinopropionate as substrates for creatine kinase. Biochem Med. 1979;21:162-167. [Medline] [Order article via Infotrieve]

9. Shoubridge EA, Jeffry FMH, Keogh JM, Radda GK, Seymour AML. CK kinetics, ATP turnover and cardiac performance in hearts depleted of creatine with the substrate analog ß-guanidinopropionic acid. Biochim Biophys Acta. 1985;847:25-32. [Medline] [Order article via Infotrieve]

10. Kapelko VI, Kupriyanov VV, Novikova NA, Lakomkin VL, Steinschneider AY, Severina MY, Veksler VI, Saks VA. The cardiac contractile failure induced by chronic creatine and phosphocreatine deficiency. J Mol Cell Cardiol. 1988;20:465-479. [Medline] [Order article via Infotrieve]

11. Zweier JL, Jacobus WE, Korecky B, Brandejs-Barry Y. Bioenergetic consequences of cardiac phosphocreatine depletion induced by creatine analogue feeding. J Biol Chem. 1991;266:20296-20304. [Abstract/Free Full Text]

12. van Deurson J, Heerschap A, Oerlemans F, Ruitenbeek W, Jap P, ter Laak H, Wieringa B. Skeletal muscles of mice deficient in muscle creatine kinase lack burst activity. Cell. 1993;74:621-631. [Medline] [Order article via Infotrieve]

13. Nascimben L, Friedrich J, Liao R, Pauletto P, Pessina AC, Ingwall JS. Enalapril treatment increases cardiac performance and energy reserve via the creatine kinase reaction in myocardium of Syrian myopathic hamsters with advanced heart failure. Circulation. 1995;91:1824-1833. [Abstract/Free Full Text]

14. Ingwall JS. Is cardiac failure a consequence of decreased energy reserve? Circulation. 1993;87(suppl VII):VII-58-VII-62.

15. Sylven C, Lin L, Jansson E, Sotonyi P, Fu L-X, Waagstein F, Hjalmarsson A, Marcus C, Bronnegard M. Ventricular adenine nucleotide translocator mRNA is upregulated in dilated cardiomyopathy. Cardiovasc Res. 1993;27:1295-1299. [Medline] [Order article via Infotrieve]

16. Rajagopalan B, Blackledge MJ, McKenna WJ, Bolas N, Radda GK. Measurement of phosphocreatine to ATP ratio in normal and diseased human heart by ³¹P magnetic resonance spectroscopy using the rotating frame-depth selection technique. Ann N Y Acad Sci. 1987;508:321-332. [Medline] [Order article via Infotrieve]

17. Conway MA, Allis J, Ouwerkerk R, Niioka T, Rajagopalan B, Radda GK. Detection of low phosphocreatine to ATP ratio in failing hypertrophied human myocardium by ³¹P magnetic resonance spectroscopy. Lancet. 1991;338:973-976. [Medline] [Order article via Infotrieve]

18. Hardy CJ, Weiss RG, Bottomley PA, Gerstenblith G. Altered myocardial high-energy phosphate metabolites in patients with dilated cardiomyopathy. Am Heart J. 1991;122:795-801. [Medline] [Order article via Infotrieve]

19. Neubauer S, Krahe T, Schindler R, Horn M, Hillenbrand H, Entzeroth C, Mader H, Kromer EP, Riegger GAJ, Lackner K, Ertl G. ³¹P magnetic resonance spectroscopy in dilated cardiomyopathy and coronary artery disease: altered high-energy phosphate metabolism in heart failure. Circulation. 1992;86:1810-1818. [Abstract/Free Full Text]

20. Mitsunami K, Okada M, Inoue T, Hachisuka M, Kinoshita M, Inuzushi T. In vivo ³¹P nuclear magnetic resonance spectroscopy in patients with old myocardial infarction. Jpn Circ J. 1992;56:614-619.[Medline] [Order article via Infotrieve]

21. Neubauer S, Horn M, Naumann A, Tian R, Hu K, Laser M, Friedrich J, Gaudron P, Schnackerz K, Ingwall JS, Ertl G. Impairment of energy metabolism in intact residual myocardium of rat hearts with chronic myocardial infarction. J Clin Invest. 1995;95:1092-1100.

22. Fitch CD, Shields RP, Payne WF, Dacus JM. Creatine metabolism in skeletal muscle, III: specificity of the creatine entry process. J Biol Chem. 1968;243:2024-2027. [Abstract/Free Full Text]

23. Jankus EF, Noren GR, Staley NA. Research note—furazolidone-induced cardiac dilation in turkeys. Avian Dis. 1972;16:958-961. [Medline] [Order article via Infotrieve]

24. Gwathmey JK. Morphological changes associated with furazolidone-induced cardiomyopathy: effects of digoxin and propranolol. J Comp Pathol. 1991;104:33-45. [Medline] [Order article via Infotrieve]

25. Hajjar RJ, Liao R, Young JB, Fuleihan F, Glass MG, Gwathmey JK. Pathophysiological and biochemical characterization of an avian model of dilated cardiomyopathy: comparison to findings in human dilated cardiomyopathy. Cardiovasc Res. 1993;27:2212-2221. [Abstract/Free Full Text]

26. Gruver EJ, Glass MG, Marsh JD, Gwathmey JK. An animal model of dilated cardiomyopathy: characterization of dihydropyridine receptors and contractile performance. Am J Physiol. 1993;265(Heart Circ Physiol 34):H1704-H1711.

27. Glass MG, Fuleihan F, Liao R, Lincoff AM, Chapados R, Hamlin R, Apstein CS, Allen PD, Ingwall JS, Hajjar RJ, Cory CR, O'Brien PJ, Gwathmey JK. Differences in cardioprotective efficacy of adrenergic receptor antagonists and Ca²⁺ channel antagonists in an animal model of dilated cardiomyopathy: effect on gross morphology, global cardiac function, and twitch force. Circ Res. 1993;73:1077-1089. [Abstract/Free Full Text]

28. Neely JR, Libermeister H, Battersby EJ, Morgan HE. Effect of pressure development on oxygen consumption by isolated rat heart. Am J Physiol. 1967;212:804-815.

29. McLeod K, Comisarow MB. Systematic errors in the discrete integration of Fourier transform nuclear magnetic resonance spectra. J Magn Reson Imaging. 1989;84:490-500.

30. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951;193:265-275. [Free Full Text]

31. Kammermeier H. Microassay of free and total creatine from tissue extracts by combination of chromatographic and fluorometric methods. Anal Biochem. 1973;56:341-345. [Medline] [Order article via Infotrieve]

32. Rosalki SB. An improved procedure for serum creatine phosphokinase determination. J Lab Clin Med. 1967;69:696-705. [Medline] [Order article via Infotrieve]

33. Bak MI, Ingwall JS. NMR-invisible ATP in heart: fact or fiction? Am J Physiol. 1992;262(Endocrinol Metab 25):E943-E947.

34. Clarke K, Anderson RE, Nedelec J-F, Foster DO, Ally A. Intracellular and extracellular spaces and the direct quantification of molar intracellular concentrations of phosphorus metabolites in the isolated rat heart using ³¹P NMR spectroscopy and phosphate markers. Magn Reson Med. 1994;32:181-188. [Medline] [Order article via Infotrieve]

35. Polimeni PI. Measurement of myocardial electrolyte distributions. Tech Life Sci Cardiovasc Physiol. 1984;317:1-34.

36. Anversa P, Beghi C, Kikkawa Y, Olivetti G. Myocardial infarction in rats: infarct size, myocyte hypertrophy, and capillary growth. Circ Res. 1986;58:26-37. [Abstract/Free Full Text]

37. Friedrich J, Nascimben L, Liao R, Ingwall JS. Phosphocreatine T₁ measurements with and without exchange in the heart. Magn Reson Med. 1993;30:45-50. [Medline] [Order article via Infotrieve]

38. Veech RL, Lawson JWR, Cornell NW, Krebs HA. Cytosolic phosphorylation potential. J Biol Chem. 1979;254:6538-6547. [Abstract/Free Full Text]

39. Forsen S, Hoffman RA. Study of moderately rapid chemical exchange reactions by means of nuclear magnetic double resonance. J Chem Phys. 1963;39:2892-2901.

40. Takaoka H, Takeuchi M, Odake M, Hayashi Y, Hata K, Mori M, Yokoyama M. Comparison of hemodynamic determinants for myocardial oxygen consumption under different contractile states in human ventricle. Circulation. 1993;87:59-69. [Abstract/Free Full Text]

41. Tian R, Gaudron P, Neubauer S, Hu K, Ertl G. Alterations of performance and oxygen utilization in chronically infarcted rat hearts. J Mol Cell Cardiol. 1996;28:321-330. [Medline] [Order article via Infotrieve]

42. Tian R, Nascimben L, Kaddurah-Daouk R, Ingwall JS. Depletion of energy reserve via the creatine kinase reaction during the evolution of heart failure in cardiomyopathic hamsters. J Mol Cell Cardiol. In press.

43. Bittl JA, Ingwall JS. Intracellular high-energy phosphate transfer in normal and hypertrophied myocardium. Circulation. 1987;75(suppl I):I-96. Abstract.

44. Friedrich J, Apstein CS, Ingwall JS. ³¹P nuclear magnetic resonance spectroscopic imaging of regions of remodeled myocardium in the infarcted rat heart. Circulation. 1995;92:3527-3538. [Abstract/Free Full Text]

45. Mirsalimi SM, Qureshi FS, Julian RJ, O'Brien PJ. Myocardial biochemical changes in furazolidone-induced cardiomyopathy of turkeys. J Comp Pathol. 1990;102:139-147. [Medline] [Order article via Infotrieve]

46. Nascimben L, Tian R, Lorell BH, Reis I, Weinberg EO, Ingwall JS. Rates of insulin-independent glucose entry and glycolysis are increased in hypertrophied hearts. Circulation. 1995;92(suppl I):I-770. Abstract.

47. Nascimben L, Pauletto P, Pessina AC, Reis I, Ingwall JS. Decreased energy reserve may cause pump failure in human dilated cardiomyopathy. Circulation. 1991;84(suppl II):II-563. Abstract.

48. Bottomley PA, Herfkens RJ, Smith LS, Bashore TM. Altered phosphate metabolism in myocardial infarction: P-31 NMR spectroscopy. Radiology. 1987;165:703-707.[Abstract/Free Full Text]

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				M. I. Miyamoto, F. del Monte, U. Schmidt, T. S. DiSalvo, Z. B. Kang, T. Matsui, J. L. Guerrero, J. K. Gwathmey, A. Rosenzweig, and R. J. Hajjar Adenoviral gene transfer of SERCA2a improves left-ventricular function in aortic-banded rats in transition to heart failure PNAS, January 18, 2000; 97(2): 793 - 798. [Abstract] [Full Text] [PDF]

				J. I. Goldhaber Sodium-Calcium Exchange : The Phantom Menace Circ. Res., November 26, 1999; 85(11): 982 - 984. [Full Text] [PDF]

				E. De Sousa, V. Veksler, A. Minajeva, A. Kaasik, P. Mateo, E. Mayoux, J. Hoerter, X. Bigard, B. Serrurier, and R. Ventura-Clapier Subcellular Creatine Kinase Alterations : Implications in Heart Failure Circ. Res., July 9, 1999; 85(1): 68 - 76. [Abstract] [Full Text] [PDF]

				M. Horn, H. Remkes, C. Dienesch, K. Hu, G. Ertl, and S. Neubauer Chronic high-dose creatine feeding does not attenuate left ventricular remodeling in rat hearts post-myocardial infarction Cardiovasc Res, July 1, 1999; 43(1): 117 - 124. [Abstract] [Full Text] [PDF]

				J. K. Gwathmey, C. S. Kim, R. J. Hajjar, F. Khan, T. G. DiSalvo, A. Matsumori, and M. R. Bristow Cellular and molecular remodeling in a heart failure model treated with the beta -blocker carteolol Am J Physiol Heart Circ Physiol, May 1, 1999; 276(5): H1678 - H1690. [Abstract] [Full Text] [PDF]

				Y. Murakami, J. Zhang, M. H. J. Eijgelshoven, W. Chen, W. C. Carlyle, Y. Zhang, G. Gong, and R. J. Bache Myocardial creatine kinase kinetics in hearts with postinfarction left ventricular remodeling Am J Physiol Heart Circ Physiol, March 1, 1999; 276(3): H892 - H900. [Abstract] [Full Text] [PDF]

				R. Tian, J. M. Halow, M. Meyer, W. H. Dillmann, V. M. Figueredo, J. S. Ingwall, and S. A. Camacho Thermodynamic limitation for Ca2+ handling contributes to decreased contractile reserve in rat hearts Am J Physiol Heart Circ Physiol, December 1, 1998; 275(6): H2064 - H2071. [Abstract] [Full Text] [PDF]

				K. W. Saupe, M. Spindler, R. Tian, and J. S. Ingwall Impaired Cardiac Energetics in Mice Lacking Muscle-Specific Isoenzymes of Creatine Kinase Circ. Res., May 4, 1998; 82(8): 898 - 907. [Abstract] [Full Text] [PDF]

				C. Baboonian and T. Treasure Meta-analysis of the association of enteroviruses with human heart disease Heart, December 1, 1997; 78(6): 539 - 543. [Abstract] [Full Text] [PDF]

				S. Neubauer, M. Horn, M. Cramer, K. Harre, J. B. Newell, W. Peters, T. Pabst, G. Ertl, D. Hahn, J. S. Ingwall, et al. Myocardial Phosphocreatine-to-ATP Ratio Is a Predictor of Mortality in Patients With Dilated Cardiomyopathy Circulation, October 7, 1997; 96(7): 2190 - 2196. [Abstract] [Full Text]

				L. Nascimben, J. S. Ingwall, P. Pauletto, J. Friedrich, J. K. Gwathmey, V. Saks, A. C. Pessina, and P.D. Allen Creatine Kinase System in Failing and Nonfailing Human Myocardium Circulation, October 15, 1996; 94(8): 1894 - 1901. [Abstract] [Full Text]

				S. Boudina, M. N. Laclau, L. Tariosse, D. Daret, G. Gouverneur, S. Bonoron-Adele, V. A. Saks, and P. Dos Santos Alteration of mitochondrial function in a model of chronic ischemia in vivo in rat heart Am J Physiol Heart Circ Physiol, March 1, 2002; 282(3): H821 - H831. [Abstract] [Full Text] [PDF]

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