INTRODUCTION

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THE FINAL REACTION of the sequence that links carbon substrate utilization to oxidative ATP synthesis is the mitochondrial ATPase (mtATPase) ATP synthase. This enzyme catalyzes the phosphorylation of ADP to form ATP. Using a porcine model of postinfarction left ventricular remodeling (LVR), we previously observed (22) that the protein levels of mitochondrial adenine translocator and F₁-ATPase were decreased in animals that developed overt congestive heart failure (CHF). On the basis of this previous finding, in the present study, we hypothesized that the decreased expression of mtATPase would require an increase in the cytosolic free-ADP level to drive mitochondrial ATP synthesis. To examine this hypothesis, we measured myocardial high-energy phosphates (HEP) with ³¹P magnetic resonance spectroscopy (MRS) in swine in which occlusion of the left circumflex coronary artery (LCx) had resulted in either compensated LVR or CHF. Measurements were obtained during basal conditions and with modest increases in cardiac work that are known not to produce HEP changes in normal hearts (4, 33). To determine whether oxygen insufficiency might contribute to HEP changes in failing hearts, myocardial deoxymyoglobin (Mb-) was assessed using ¹H MRS. Mitochondrial protein levels of the F₀F₁-ATPase subunits were examined by Western blotting.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

All experimental procedures were performed in accordance with the animal use guidelines of the University of Minnesota, and the experimental protocol was approved by the University of Minnesota Research Animal Resources Committee. The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1985).

Infarct production by coronary ligation. Details of the animal model of postinfarction LVR were as described previously (20, 37). In brief, Young Yorkshire swine (age 45 days; weight ~10 kg) were anesthetized with pentobarbital sodium (30 mg/kg iv), intubated, and ventilated with a respirator and supplemental oxygen. Arterial blood gases were maintained within the physiological range by adjusting the respiratory settings and the oxygen flow. A left thoracotomy was performed, and 0.5 cm of the proximal LCx was dissected free and completely occluded with a ligature. The animals were then observed in the open-chest state for 60 min. When ventricular fibrillation occurred, electrical defibrillation was performed immediately. This procedure was usually successful. The chest was then closed; if the heart was dilated, the pericardium was left open. The animals were given standard postoperative care including analgesia until they ate normally and were active. LCx occlusion was performed in 41 pigs; of these, 4 pigs died suddenly during the first 24 h after LCx ligation surgery. Studies were performed ~6 wk after the LCx occlusion. Among the 37 pigs, 8 developed CHF as evidenced by cyanosis or ascites. These 8 animals formed the CHF group. Of the remaining 29 pigs, 9 with LCx ligation formed the LVR group. The remaining 20 pigs with compensated LVR underwent experiment protocols for other studies and therefore were not included in this study.

Surgical preparation. Before surgery, 17 animals with LVR and 7 size-matched normal animals were anesthetized with -chloralose (100 mg/kg then 20 mg · kg¹ · h¹ iv), intubated, and ventilated with a respirator and supplemental oxygen. Arterial blood gases were maintained within the physiological range by adjusting the respiratory settings and oxygen flow. A heparin-filled polyvinyl chloride catheter (3.0 mm OD) was introduced into the right femoral artery and advanced into the ascending aorta. A sternotomy was performed and the heart was suspended in a pericardial cradle. A second heparin-filled catheter was introduced into the left ventricle through the apical dimple and secured with a purse-string suture. A similar catheter was inserted into the left atrium through the atrial appendage. A bipolar pacing electrode was sutured onto the right atrial appendage. A 25-mm-diameter NMR surface coil was sutured onto the left ventricular (LV) anterior wall; the infarct region was avoided. The pericardial cradle was then released, and the heart was allowed to assume its normal position in the chest. The surface coil leads were connected to a balanced and tuned external circuit. The animals were then placed in a Lucite cradle and positioned within the magnet.

Spatially localized ³¹P NMR spectroscopic technique. Spatially localized ³¹P NMR spectroscopy was performed using adiabatic plane rotation pulses for phase modulation (RAPP) in image-selected in vivo spectroscopy (ISIS) (11, 24, 33). Detailed experiments documenting the voxel profiles and volumes and the spatial resolution attained by this method have been published previously (11, 24, 33). In this application of the RAPP-ISIS technique, signal origin was first restricted to an 18 × 18-mm two-dimensional column perpendicular to the LV wall and further localized in three well-resolved and five partially resolved layers along the column and hence across the LV wall. Localization along the column was based on magnetic field strength generated at the center of the surface coil (B₁); the phase encoding employed a nine-term Fourier series window as previously described (33). Complete transmural data sets were obtained in 10-min time blocks using a repetition time of 6-7 s, which allows full relaxation for ATP and P_i and ~90% relaxation of the phosphocreatine (PCr) resonance. Whole wall spectra were obtained with the ISIS, which defined a column that was 2.3 × 2.3 cm² and perpendicular to the heart wall. NMR data acquisition was gated to the cardiac and respiratory cycles by using the cardiac cycle as the master clock to drive both the respirator and the spectrometer as previously described (24). The surface coil was constructed of a single-turn copper wire 28 mm in diameter with each side of the coil leads soldered to a 33-pF capacitor. Calibration was facilitated by including a polyethylene capillary filled with 15 µl of 3 M phosphonoacetic acid within the inner diameter of the coil. The ratio of PCr to ATP was calculated for each spectrum. The integral value of the basal P_i (for most of the hearts, this value was zero) was subtracted from the P_i values acquired during subsequent experimental conditions to obtain P_i values. P_i data are presented as P_i/PCr. All resonance intensities were quantified using integration routines provided by SISCO software.

Calculation of myocardial-free ADP level. The myocardial- free ADP levels were calculated from the creatine kinase (CK) equilibrium expression (17) using an equilibrium constant (K_eq) of 1.66 × 10⁹ and a cytosolic pH of 7.1: [ADP] = ([ATP] [CR_free])/([PCr] [H⁺] K_eq), where square brackets indicate concentrations. PCr and ATP values were obtained from the spectra calibrated by the biopsy-measured ATP levels. Free creatine was calculated by subtracting the PCr values from the biopsy-obtained measurements of total creatine.

¹H NMR spectroscopic technique. We have recently reported the ¹H NMR methods in detail elsewhere (6, 20). In brief, radio frequency transmission and signal detection were performed with the dually tuned 28-mm-diameter surface coil. A single-pulse collection sequence with a frequency-selective Gauss excitation pulse (1 ms) was used to selectively excite the N- proton signal of proximal histidine in deoxymyoglobin (Mb-) resonance. This provided sufficient water suppression because of the large chemical-shift difference between water and Mb- (>14 kHz), and other techniques such as the Cornell High-Energy Synchrotron Source [CHESS (10)] and an inversion-recovery pulse did not significantly improve water suppression. The NMR signal was optimized by adjusting the radio frequency pulse power using the water signal as a reference. A short repetition time (TR) of 25 ms was used owing to the short T1 of Mb-. Each spectrum was acquired in 5 min (10,000 free induction decays). Although the short T1 of Mb- and the fast acquisition prevented gating to the cardiac cycle, the signal loss due to motion was negligible because of the inherently broad line width of the Mb- peak.

Hemodynamic measurements. Aortic and LV pressures were measured using Spectromed TNF-R pressure transducers positioned at midchest level. All data were recorded on an eight-channel Coulbourne R14-28 direct-writing recorder.

Myocardial blood flow measurements. Myocardial blood flow (_m) was measured using 15-µm-diameter radionuclide-labeled microspheres as previously described (3, 20, 33). Microspheres labeled with four different radioisotopes (⁵¹Cr, ⁸⁵Sr, ⁹⁵Nb, and ⁴⁶Sc) were agitated in an ultrasonic mixer for 10 min before injection. A suspension containing 3 × l0⁶ microspheres was injected through the left atrial catheter while a reference sample of arterial blood was drawn from the aortic catheter at a rate of 15 ml/min. Radioactivity levels in the myocardial and blood reference specimens (C_m and C_r, respectively) were determined using a gamma spectrometer (Cobra; Packard Instruments; Downers Grove, IL). Knowing the rate of withdrawal of the reference blood specimen (_r) and C_r, the C_m was used to compute _m as: _m = _r (C_m/C_r).

mtATPase subunit protein levels. Frozen heart samples (weight ~100 mg) were powdered in a liquid nitrogen-cooled mortar and then added to 1 ml of ice-cold buffer (containing 0.25 M sucrose, 50 mM Tris · HCl, and 10 mM sodium azide at pH 8.5). The samples were homogenized and then incubated for 1 h at room temperature. All tissues were centrifuged at 10,000 g for 10 min at room temperature. The supernatants were used for Western blotting, and protein concentration was determined by a modified Lowry analysis (protein assay kit; Sigma). The antibodies 7B3 (against -subunit F₁- ATPase), 19D3 (against -subunit F₁-ATPase), 2B1B1 [against the oligomycin sensitivity-conferring protein (OSCP) subunit], and 2H10 [against the initiation factor 1 (IF₁) subunit] were produced as described (2, 18). Total protein extract (15 µg) was loaded onto a standard discontinuous 12% SDS polyacrylamide gel run at 200 V for 90 min. Proteins were transferred to nitrocellulose membranes (Bio-Rad). Transfer efficiency was assessed by staining the gel with Coomassie blue before and after transfer. Nonspecific binding sites were blocked by incubating the membrane overnight in 5% nonfat milk buffer. The primary antibody was diluted in antibody buffer [1% nonfat milk in 0.1% Tween 20-Tris-buffered saline (TTBS)] and incubated with the membranes for 2 h on a rotating cylinder. The membrane was washed three times with TTBS buffer for 30 min. Appropriate horseradish peroxidase-labeled secondary antibodies (goat anti-mouse) were then incubated with the membrane for 1 h. The membrane was again washed three times in TTBS buffer before undergoing a 1-min incubation with enhanced chemiluminescent substrate (Amersham). Light emission was detected by exposure to Kodak RX autoradiography film. Signal densities were quantified by a laser densitometric scanner (Bio-Rad) and were normalized to the intensities of the same amount of -actin protein.

Study protocol. Ventilation rate, volume, and inspired oxygen content were adjusted to maintain physiological values for arterial PO₂, PCO₂, and pH. Aortic and LV pressures were monitored continuously throughout the study. _m and hemodynamic measurements were acquired simultaneously with the acquisition of ¹H and ³¹P magnetic resonance spectra. After baseline data were obtained, dobutamine and dopamine (Db and Dp, respectively) were simultaneously infused (2.5-10 µg · kg¹ · min¹ iv each) to increase the cardiac work state to a rate pressure product (RPP) of ~20,000 mmHg · beats¹ · min¹. In failing hearts, if the RPP did not reach 20,000 mmHg/min in response to catecholamine stimulation, atrial pacing at a rate of 200 beats/min was then started. A steady state was achieved after ~10 min, and all measurements were repeated. After completion of this protocol, in four normal pigs and four animals with LCx ligation (two with CHF), the left anterior descending artery or the second diagonal branch were completely occluded, and Mb- measurements were repeated. The hearts were then rapidly removed and prepared for blood flow measurements and tissue sampling.

Tissue preparation. Once the study was completed, a myocardial drill biopsy of the LV wall was taken and frozen in liquid nitrogen for subsequent analysis of ATP and total creatine contents using an HPLC technique (26). The heart was then fixed in 10% buffered formalin. The region of myocardium directly beneath the surface coil was removed and sectioned into three transmural layers from epicardium to endocardium, weighed on an analytic balance, and placed into vials for counting. Similar myocardial specimens were obtained from the lateral and posterior LV walls to ensure that the measurements from the region of myocardium corresponding to the surface coil were typical for the entire left ventricle.

Data analysis. Hemodynamic data were measured from the strip-chart recordings. Transmural blood flow distribution was determined from the microsphere measurements. Data were analyzed with one-way ANOVA for repeated measurements. A value of P < 0.05 was considered significant. When a significant result was found, individual comparisons were made using Scheffé's method.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Anatomic data. The anatomic data are summarized in Table 1. In the nine LVR swine, the LV weight-to-body weight ratio (LVW/BW) was increased by 58% compared with seven size-matched normal swine (P < 0.05). The eight CHF swine demonstrated a greater degree of hypertrophy, with the LVW/BW ratio increased by 86% (P < 0.05). The right ventricular weight-to-body weight ratio was also increased in hearts with postinfarction LVR, but only in the hearts with CHF did this difference reach statistical significance (see Table 1; P < 0.01).

Hemodynamic data. Hemodynamic data are shown in Table 2. None of the hemodynamic variables were significantly different between the normal and LVR groups either at baseline or during the increased cardiac work state (see Table 2). In contrast, in hearts with CHF, the aortic and LV systolic pressures were significantly lower, and the LV end-diastolic pressure was significantly higher during the basal state (see Table 2; P < 0.05). Catecholamine stimulation induced comparable increases of the RPP in the normal and LVR groups (see Table 2); however, the CHF hearts required a high dose of catecholamine (Dp and Db, 10 µg · kg¹ · min¹ iv each) compared with normal animals and pigs with LVR (Dp and Db, 2.5-10 µg · kg¹ · min¹ iv each). In addition, four of the seven animals with CHF required pacing at 200 beats/min to reach the desired increase in cardiac work state. These data are consistent with the previous observations that hearts with CHF have decreased catecholamine responsiveness (31).

Myocardial blood flow. Mean _m measurements were not significantly different among the three groups of animals under basal conditions, and values increased similarly during the increased cardiac work state (see Table 3). The inner blood flow-to-outer blood flow ratio (Endo/Epi) tended to be lower in the CHF animals than in the other two groups, both during basal conditions and during catecholamine infusion, but this difference did not achieve statistical significance (see Table 3).

Transmural HEP and P_i levels. Myocardial HEP and P_i data are summarized in Table 4. The voxel-labeled Epi was positioned over the outer edge of the LV wall, the voxel-labeled Endo was over the subendocardium (most distant from the coil), and the voxel-labeled Mid was over the midwall. Spectra obtained at baseline were characterized by high PCr and ATP levels, whereas P_i was too low to be detected. At baseline, the PCr-to-ATP (PCR/ATP) ratios were significantly lower in the subendocardium than in the subepicardium in both normal hearts and hearts with compensated LVR (see Table 4). Under basal conditions, the PCr/ATP was significantly lower in Endo than Epi in both normal and LVR hearts, but not in hearts with CHF (see Table 4). During basal conditions, the PCr/ATP was significantly lower than normal in the Mid and Endo layers of hearts with LVR. The PCr/ATP was decreased further in animals with CHF, and was significantly lower than in normal hearts and LVR hearts in all transmural layers during basal conditions (see Table 4). Catecholamine infusion caused no change in the PCr/ATP in any transmural layer of normal or LVR hearts. In contrast, catecholamine infusion caused a further significant decrease of the PCr/ATP in all transmural layers of hearts with CHF and was associated with the appearance of a P_i resonance (see Table 4).

Biopsy data and myocardial free ADP concentration. Biopsy data are summarized in Table 5. ATP levels were significantly lower in both LVR and CHF groups compared with normal hearts (see Table 5). Myocardial total creatine levels tended to be lower in both groups of hearts with LVR, but this was significant only in the hearts with CHF (P < 0.05; Table 5). Calculated whole wall free ADP levels were significantly increased in both groups of remodeled hearts, and this was most prominent in hearts with CHF (see Table 5). Calculated free ADP levels did not change during catecholamine infusion in normal or LVR hearts. In contrast, catecholamine infusion caused a significant further increase of calculated free ADP in hearts with CHF (see Table 5).

Myoglobin saturation. No Mb- resonance was detected in any heart of any group under basal conditions or during the increased cardiac work state. During coronary artery occlusion (which was done as a positive control in four normal hearts, two hearts with LVR, and two hearts with CHF to verify that the Mb- resonance could be detected), a prominent Mb- resonance appeared at 71 ppm upfield of the water resonance.

Mitochondrial protein levels of mtATPase subunits. Western blots of mitochondrial -F₁-ATPase, OSCP, and IF₁ subunits are shown in Figs. 1-3, respectively. Mean data normalized to -actin are summarized in Fig. 4. In hearts with compensated LVR, mtATPase protein subunit expression was not significantly different from normal (P = not significant; Figs. 1-4). Compared with normal hearts, in hearts with CHF, mitochondrial -F₁-ATPase was decreased by 36% (P < 0.05), -F₁-ATPase was decreased by 16% (P < 0.05), OSCP was decreased by 40% (P < 0.01), and IF₁ was decreased by 41% (P < 0.01).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Representative Western blots showing the -subunit F1-ATPase protein migrating to the position corresponding to ~50 kDa on 12% SDS-polyacrylamide gel. The numbers 1, 2, 3, and 4 represent individual animals in the respective groups. Densitometry of the autoradiograms showed that protein levels were decreased significantly in the failing hearts. LVR, left ventricular remodeling; CHF, congestive heart failure.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Representative Western blots showing the oligomycin sensitivity-conferring protein (OSCP) subunit migrating as a protein of ~20 kDa. Densitometry of the autoradiograms showed that OSCP protein subunit levels were significantly decreased in the CHF group.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Representative Western blots showing the initiation factor 1 (IF1) subunit, which was significantly decreased in hearts with CHF.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Densitometric intensities for protein bands from Western blots of -, -, OSCP, and IF1 ATPase subunits normalized to -actin. Values are means ± SE. Normal, n = 7; LVR, n = 8; CHF, n = 8 pigs. *P < 0.05 vs. normal; **P < 0.01 vs. normal. Levels of -, -, OSCP, and IF1 mitochondrial ATPase subunits were decreased significantly only in hearts with CHF.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The present experimental model of coronary artery occlusion is characterized by prominent remodeling of the noninfarcted myocardium with dilatation of the LV chamber and elongation and hypertrophy of individual myocytes (37). Alterations of HEP content and the regulation of myocardial oxidative phosphorylation are also present; these changes are most prominent in animals that develop overt CHF (20, 37). Despite the presence of significant remodeling, hearts that remain compensated retain normal responses to the stress of rapid pacing or catecholamine infusion (20). Because failing hearts have a blunted response to catecholamines (20, 31), in the present study, atrial pacing was combined with catecholamine infusion to produce a modest increase in cardiac workload with RPPs approaching 20,000 mmHg · beats¹ · min¹. In normal hearts and hearts with compensated LVR, increases in cardiac work of this magnitude occurred with no change of HEP levels and without detectable P_i (see Table 4 ans Refs. 4, 20, 23, 33). Only when RPPs exceed ~45,000 mmHg · beats¹ · min¹ are reductions of PCr and accumulation of P_i observed in normal hearts (33). However, in the animals with CHF, even the modest increase of RPP to ~20,000 mmHg · beats¹ · min¹ achieved in the present study resulted in a further decrease of the PCr/ATP with the appearance of P_i and an increase in calculated myocardial free ADP. Although the HEP changes that occurred during the increased work state in the hearts with CHF were similar to those observed during myocardial ischemia, Mb- was not detected. The ability to assess myocardial myoglobin oxygenation allows us to conclude that oxygen insufficiency cannot account for either the HEP alterations present at baseline or the further perturbation of HEP levels during the increased cardiac work state in the failing hearts.

In agreement with previous reports (27, 37), myocardial ATP concentrations were decreased in failing hearts in the present study. It is possible that this abnormality is related to the increased ADP levels in the failing hearts. An increase of myocardial free ADP would activate adenylate kinase (myokinase), which catalyzes the transfer of a phosphoryl group between two molecules of ADP to form one molecule each of AMP and ATP (15, 16). AMP can be acted upon by nucleotide phosphorylase to produce adenosine (28). Unlike the nucleotides, adenosine can cross the cell membrane into the interstitial space where it is further degraded to inosine and hypoxanthine to leave the heart through the coronary circulation (13). Even in the normal heart, increases of cardiac work produced by norepinephrine infusion are associated with increases of adenosine released into intracoronary venous blood (9). The greater increases of myocardial ADP levels during catecholamine infusion in the hypertrophied hearts in the present study might be expected to further augment adenosine loss from the heart. Loss of the total adenine nucleotide pool could result in a reduction of ATP, because the de novo resynthesis of adenine nucleotide is a slow and energy-costly process, where inosine monophosphate is produced from ribose-5-phosphate utilizing six HEP bonds (19). For example, the ATP depletion that occurs in postischemic stunned myocardium requires several days for recovery (29, 37). Loss of the adenine nucleotide pool in the failing heart may be facilitated by the persistently normal blood flow levels. In contrast to ischemia, where coronary blood flow is reduced, the normal coronary flow rates in the failing hearts might promote washout of adenosine from the heart.

A central question is whether the observed HEP abnormalities might contribute to contractile dysfunction in the failing hearts. Although ATP levels were decreased in the failing hearts, the myocardial ATP concentrations were nevertheless many times greater than the Michaelis-Menten (K_m) value of either myosin ATPase or the sarcoplasmic(endo)reticulum calcium ATPase (SERCA) (7, 14). Furthermore, in postischemic stunned hearts, ATP levels can be more severely depressed than in the failing hearts of the present study, but contractile function improves in response to catecholamine administration, which demonstrates that the decreased ATP level does not prevent normal contractile performance (1). Consequently, it is unlikely that the decrease of ATP in the failing hearts in the present study can account for the depressed contractile performance. A decrease of the ATP-to-ADP ratio, as seen in the failing hearts, does result in decreased free energy released per unit of ATP hydrolysis (G) (14, 30). Reductions of G have the potential to decrease LV contractile performance but only at values substantially below those calculated in the failing hearts in the present study (30).

Mitochondrial ATPase. Mitochondrial ATP synthase embedded in the mitochondrial inner membrane drives ATP synthesis using energy generated from the electrochemical proton gradient across the inner mitochondrial membrane (5). The - and -subunits are homologous; both bind nucleotides, but only the -subunit has catalytic activity. The OSCP is involved in the efficient binding of F₁ to F₀. The ATPase inhibitor protein, IF₁, is a small basic protein that can protect ATP from hydrolysis by the F₁-ATPase. In the present study, the -, -, OSCP, and IF₁ subunits were all lower in failing hearts than in normal hearts, and the decreases were significantly related to the degree of reduction of the myocardial PCr/ATP, which implies that reductions of ATPase protein expression were associated with the degree of increase in ADP levels. A decrease in the -F₁-ATPase activity or an increase in its apparent K_m value with respect to ADP could require an increase in cytosolic ADP and P_i levels to maintain a given rate of ATP synthesis. However, because of the presence of endogenous inhibitors including IF₁, ATP synthetic activity cannot be directly equated with the myocardial F₁-ATPase content. Thus Scholz and Balaban (25) found that incubation of normal myocardium in high-salt-pH buffer, which is known to promote release of IF₁ from F₁-ATPase, caused an increase in ATPase activity. This demonstrates baseline inhibition and indicates that protein content cannot be directly equated with in vivo enzyme activity. The investigators speculated that the baseline inhibition likely resulted from binding of IF₁ (or possibly the calcium-binding inhibitor protein) (32) to the F₁-ATPase. ATPase activity was not measured in the present study, but it is likely that the decreased protein expression could limit the maximum ATP synthesis rates achievable during high work states in the failing hearts. It is tempting to speculate that the observed decreases of mitochondrial ATP synthase proteins could contribute to a compensatory increase of cytosolic ADP levels (and therefore the decreased PCr/ATP) to maintain ATP synthesis rates in the failing hearts. However, the present data cannot prove a causal relationship between the observed decrease of ATPase protein subunits and the increased levels of ADP and P_i in the failing hearts.

Limitations. To compensate for the blunted response of the failing hearts to catecholamine stimulation, rapid pacing was used in four of the seven failing hearts. It could be argued that in these hearts the increase of P_i and decrease of the PCr/ATP were caused by ischemia secondary to tachycardia. Thus in hearts with left ventricular hypertrophy secondary to chronic pressure overload, we found that pacing-induced tachycardia (without catecholamine infusion) resulted in a decrease of myocardial HEP levels that was most severe in the subendocardium (34). These HEP changes were caused by a redistribution of blood flow away from the inner layer of the LV wall during tachycardia (34). In contrast, in normal hearts or hearts with eccentric hypertrophy secondary to mitral regurgitation, pacing caused an increase of blood flow that was uniform across the LV wall, and myocardial HEP levels remained constant (35). Furthermore, using the present model of postinfarction LVR, we previously observed that coronary flow reserve during maximum vasodilation with adenosine was normal in the failing hearts (37). Myocardial oxygen consumption was not measured in the present study because the CHF hearts had difficulty tolerating the additional surgical manipulation required to insert a coronary venous catheter. Nevertheless, the normal response of _m to the increased workload in the failing hearts, as well as the lack of detectable Mb-, indicates that the HEP changes observed during the increased workload in the present study were not caused by ischemia.