Cardiovascular Research

Cardiovascular Research 1999 42(3):616-626; doi:10.1016/S0008-6363(98)00332-0
© 1999 by European Society of Cardiology

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Copyright © 1999, European Society of Cardiology

Myocardial oxygenation at high workstates in hearts with left ventricular hypertrophy

Robert J. Bache^*, Jianyi Zhang, Yo Murakami, Yi Zhang, Yong K. Cho, Helmut Merkle, Guangrong Gong, Arthur H.L. From and Kamil Ugurbil

Cardiovascular Division, Department of Medicine, University of Minnesota Medical School, Box 508, UMHC, Minneapolis, MN 55455, USA

^* Corresponding author. Tel.: +1-612-625-2454; Fax: +1-612-626-4411. E-mail address: bache001@maroon.tc.umn.edu (R.J. Bache)

Received 11 June 1998; accepted 30 October 1998

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
Background: High cardiac workloads produced by catecholamine infusion result in loss of myocardial phosphocreatine (PCr) and accumulation of inorganic phosphate (Pi) which are more prominent in hearts with left ventricular hypertrophy (LVH) than in normal hearts. Since ischemia can cause changes in phosphorylated compounds similar to those during catecholamine stimulation, this study tested the hypothesis that the exaggerated depletion of PCr and accumulation of Pi during high workloads in LVH is the result of impaired myocyte oxygenation. Methods and results: ³¹P- and ¹H-NMR spectroscopy were used to determine myocardial high energy phosphate levels and myoglobin desaturation, respectively, in eight normal dogs and nine dogs with LVH produced by ascending aortic banding. The mean LV weight/body weight ratio was approximately twice normal in the LVH group. Infusion of dobutamine (15 and 30 µg/kg/min), and dobutamine+dopamine (each 20 µg/kg/min) caused progressive similar increases in the heart ratexsystolic LV pressure product to a maximum of 57.4±3.3·10³ in normal and 63.9±2.7·10³ in LVH animals, while myocardial oxygen consumption increased from 0.09±0.01 to 0.24±0.04 in normals and from 0.10±0.02 to 0.25±0.03 ml/min/g in LVH. PCr/ATP ratios during basal conditions were lower in LVH hearts (1.73±0.10, 1.61±0.09 and 1.51±0.09 in subepicardium, midwall and subendocardium, respectively) as compared with normals (2.24±0.09, 2.01±0.08 and 1.89±0.07; each p<0.01 normal vs. LVH). Catecholamine infusions caused dose-related decreases in PCr/ATP and appearance of Pi which was more marked in LVH than in normal hearts. ¹H-NMR spectroscopy did not detect deoxymyoglobin in either normal or LVH hearts even during the highest workloads. In contrast, occlusion of the anterior descending coronary artery resulted in a large deoxymyoglobin signal. Conclusions: Increases of cardiac work produced by catecholamine stimulation resulted in greater decreases of PCr and greater increases of Pi in hypertrophied than in normal hearts. These abnormalities were not the result of inadequate intracellular oxygen availability and consequently cannot be ascribed to demand ischemia.

KEYWORDS High energy phosphates; Deoxymyoglobin; Nuclear magnetic resonance; Phosphocreatine; Dog

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See Editorial of this article by C. Gibbs (pages 567–570) in this issue.

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
In hearts with left ventricular hypertrophy (LVH) secondary to ascending aortic banding the inotropic and chronotropic stimulation produced by infusion of dobutamine resulted in loss of phosphocreatine (PCr) and accumulation of inorganic phosphate (Pi) which was significantly more prominent than in normal hearts [1]. Although these high energy phosphate (HEP) changes would be compatible with demand-induced ischemia, coronary vasodilator reserve was not exhausted during dobutamine infusion, so that myocardial blood flow-rates could have increased if myocardial metabolic needs were not met [1]. An alternate possibility for the observed HEP changes during dobutamine infusion is that oxygen delivery is limited by impaired diffusion. Intercapillary distances have been reported to be increased in the pressure overloaded hypertrophied LV, since the capillary:myocyte ratio remains unchanged as myocyte diameter increases during the hypertrophic process [2, 3]. As a result, the average intercapillary distance is increased so that the mean distance for oxygen diffusion is greater than normal in the hypertrophied heart. In this situation oxygen deficiency at the mitochondrial level might occur as the result of impaired diffusion with no abnormality in blood flow-rates.

The degree of myoglobin deoxygenation in myocytes can be quantitated with ¹H-NMR and used to determine intracellular oxygen tension. The unpaired electron spin in the heme-Fe(II) complex of deoxymyoglobin (Mb-) extends over the proximal histidyl N proton signal detected by ¹H-NMR spectroscopy [4]. Jue and associates [5, 6] have demonstrated that graded reductions of coronary perfusion in isolated rat hearts which produced decreases in PCr were associated with reciprocal increases in Mb- detected with ¹H-NMR. In a recent ¹H-NMR study in normal dogs we observed that decreases of myocardial blood flow produced by a graded coronary artery stenosis resulted in increasing Mb- signal which was proportional to the decrease in blood flow [7], thus demonstrating the feasibility of this methodology for assessment of myocardial oxygenation in vivo. This technique was used in the present study to test the hypothesis that the HEP changes observed during high workstates produced by catecholamine administration in the hypertrophied heart are the result of inadequate oxygen availability. Transmurally localized ³¹P-NMR spectroscopy was combined with ¹H-NMR for detection of Mb- to determine whether alterations in HEP during the high workstates produced by dobutamine administration are the result of decreased intracellular oxygen availability to the mitochondria.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985) and the protocol was approved by the Animal Care Committee of the University of Minnesota.

2.1 Production of LVH
Nine mongrel dogs 8 weeks of age were anesthetized with sodium pentobarbital (25–30 mg/kg i.v.), intubated and ventilated with a respirator. A right thoracotomy was performed in the third intercostal space and the ascending aorta, approximately 1.5 cm above the aortic valve, was mobilized and encircled with a polyethylene band 2.5 mm in width [8]. While simultaneously measuring LV and distal aortic pressures, the band was tightened until a 20–30 mmHg peak systolic pressure gradient was achieved across the narrowing. The chest was then closed, the pneumothorax evacuated, and the animals allowed to recover. LVH occurred progressively as the area of aortic constriction remained fixed in the face of normal body growth. At approximately 1 year of age, animals were returned to the laboratory for study.

2.2 Experimental preparation
The nine animals with LVH, as well as eight normal dogs that were used as a control group, were premedicated with morphine sulfate (1 mg/kg s.c.) and anesthetized with -chloralose (100 mg/kg i.v. followed by an infusion of 10 mg/kg per h). Animals were intubated and ventilated with a respirator with supplemental oxygen; the ventilator rate and volume as well as the inspired O₂ content, were adjusted to maintain arterial blood gases and pH within the physiologic range. A polyvinyl chloride catheter (3.0 mm O.D.) filled with heparin–saline was introduced into the right femoral artery and advanced into the ascending aorta. A left thoracotomy was performed in the fifth intercostal space and the heart suspended in a pericardial cradle. A heparin–saline filled catheter was introduced into the LV through the apical dimple and secured with a purse-string suture. A similar catheter was placed into the left atrium through the atrial appendage. A hydraulic occluder was placed around the left anterior descending coronary artery (LAD). An NMR surface coil was sutured to the anterior LV wall overlying the region perfused by the LAD. The surface coil was constructed of a single turn of copper wire and incorporated a 33 pF capacitor; surface coils for normal and LVH hearts were 28 and 35 mm in diameter, respectively. The surface coil leads were connected to a balanced-tuned circuit external and perpendicular to the thoracotomy incision. The pericardial cradle was released and the heart allowed to assume its normal position. The animals were then placed in a Lucite cradle and positioned within the magnet.

2.3 Myocardial blood flow
Myocardial blood flow was measured with microspheres, 15 micron in diameter, labeled with ¹⁴¹Ce, ⁵¹Cr, ⁹⁵Nb, ⁸⁵Sr or ⁴⁶Sc (NEN, Boston, MA, USA). Microspheres were agitated in an ultrasonic mixer for 15 min before injection. For each measurement 3·10⁶ microspheres were administered into the left atrial catheter and flushed with 5 ml of normal saline. To provide a calibration for the blood flow determinations, a reference sample of arterial blood was withdrawn from the aortic catheter using a roller pump at a rate of 15 ml/min beginning 5 s before the microsphere injection and continuing for 120 s.

2.4 ³¹P-NMR spectroscopic technique
Measurements were performed in a 40 cm bore 4.7 Tesla magnet interfaced with a Spectroscopy Imaging Systems (Fremont, CA, USA) computer console. Radio-frequency (RF) transmission and signal detection were performed with a surface coil dually tuned for ³¹P and ¹H; resonant frequencies were 81 and 200.1 MHz, respectively. A capillary containing 15 µl of 3 M/l phosphonoacetic acid was placed at the coil center to serve as a reference [9]. The proton signal from water detected with the surface coil was used to homogenize the magnetic field and to adjust the position of the animal in the magnet so that the coil was at or near the magnet and gradient isocenters. This was accomplished using a spin–echo experiment and a readout gradient [9]. The information gathered in this step was also utilized to determine the spatial coordinates for spectroscopic localization. Using B_o (static magnetic field) gradients and adiabatic inversion pulses, signal origin was restricted to a column coaxial with the surface coil and perpendicular to the LV wall; the column dimensions were 23 mmx23 mm in LVH hearts and 18 mmx18 mm in normal hearts. Within this column, the signal was further localized to 5 voxels across the LV wall from epicardium to endocardium using the B₁ (RF magnetic field) gradient centered about 45°, 60°, 90°, 120° and 135° phase angles [9]. The details of the adiabatic inversion pulses, the plane rotation adiabatic BIR-4 pulse, the Fourier coefficients, and the multiplication factors employed to construct the voxels have been reported elsewhere [9]. Using the B₁ gradient for localization along the coil axis, an increase in phase angle shifts the voxel further from the surface coil (i.e. "deeper" into the LV muscle). Because of the nonlinear nature of the B₁ gradient, voxel width is largest for the 45° ("deepest") voxel; this voxel is centered approximately one radius distance from the coil with most signal contained between 0.8 and 1.2 radius distance. Despite the nonuniform voxel volume, the detected signal per unit spins is nearly uniform between voxels (less than 20% variation) because the decreasing sensitivity with increasing distance from the coil compensates for the increasing voxel volume [9]. For this reason, intensities between voxels can be compared directly, except for the innermost voxel where the metabolite content may be underestimated due to a partial volume effect (i.e. the voxel may not contain exclusively subendocardial muscle but may be partially occupied by blood in the LV chamber). However, since the partial volume phenomenon affects all phosphorylated compounds equally, ratios of these compounds (i.e. PCr/ATP; Pi/PCr) are not altered.

The LV pressure signal was used to gate NMR data acquisition to the cardiac cycle while respiratory gating was achieved by triggering the ventilator to the cardiac cycle between data acquisitions [9]. Spectra were recorded in late diastole with a pulse repetition time of 6–7 s. This repetition time allowed full relaxation for ATP and Pi resonances, and approximately 90% relaxation for the PCr resonances. PCr resonance intensities were corrected for this saturation; the correction factor was determined for each heart from two spectra recorded consecutively without transmural differentiation, one with an 18 s repetition time to allow full relaxation and the other with the 6–7 s repetition time used during the study. Each set of spatially localized spectra consisted of 96 scans accumulated in a 10 min block of time.

Chemical shifts were measured relative to PCr which was assigned a chemical shift of –2.55 ppm relative to 85% phosphoric acid.

2.5 1H-NMR spectroscopic technique
RF transmission and signal detection were performed with the previously described surface coils for normal or LVH hearts. A single pulse-collection sequence with a frequency selective Gauss excitation pulse (1 ms) was used to selectively excite the N proton signal of the proximal histidine of Mb- [7]. This provided sufficient water suppression due to the large chemical shift difference between water and Mb (>14 kHz) and other techniques such as chemical shift selective pulse (CHESS) and inversion recovery pulse did not significantly improve water suppression. The NMR signal was optimized by adjusting the RF pulse power using the water signal as a reference. A short repetition time (TR=25 ms) was used due to short T₁ of Mb-. Each spectrum was acquired in 5 min (10 000 free induction decays). Although the short T₁ of Mb- and fast acquisition prevent gating to the cardiac cycle, the signal loss due to motion is negligible due to the inherently broad line width of the Mb- peak. Resonance intensities were quantified using integration routines provided by Varian Corp., Palo Alto, CA [9].

2.6 Hemodynamic measurements
Aortic and LV pressures were monitored with fluid filled pressure transducers (Spectramed) positioned at midchest level. Data were recorded on an eight-channel direct writing recorder (Coulbourne Instrument Company, Lehigh Valley, PA, USA). LV pressure was recorded at normal and high gain for measurement of end-diastolic pressure. Hemodynamic data were recorded continuously throughout the study.

2.7 Oxygen consumption measurements
Blood specimens were withdrawn anaerobically into iced syringes from the aortic and coronary sinus catheters (3 ml each). PO₂, PCO₂ and pH were measured with a blood gas analyzer (model 1304, Instrumentation Laboratory, Lexington, MA, USA) calibrated with known gas mixtures. Hemoglobin (Hb) content was determined by the cyanomethemoglobin method. Blood oxygen content was calculated as Hbx1.34 percent O₂ saturation+(0.0031xPO₂), using the oxygen dissociation curve for canine blood [10]. Myocardial oxygen consumption (MVO₂) was computed as the product of myocardial blood flow and the oxygen content difference between aortic and coronary venous blood.

2.8 Study protocol
Arterial blood gases and pH were monitored throughout the study and the ventilator volume and inspired O₂ content were adjusted to maintain physiologic values. Baseline hemodynamic data, Mb- data (¹H-NMR), and ³¹P-NMR spectra were obtained over a 20 min period. Heparinized arterial and coronary venous blood samples were collected anaerobically midway through each acquisition period for determination of PO₂, PCO₂ and pH. Microspheres were injected at the midpoint of each NMR data acquisition period for determination of myocardial blood flow. After baseline measurements were acquired, an intravenous infusion of dobutamine at a dose of 15 µg/kg/min was started. After hemodynamic values had stabilized (approximately 5 min), all measurements were repeated. The dobutamine infusion was then increased to 30 µg/kg/min i.v. and all measurements were repeated in the ensuring 20 min. Finally, all measurements were repeated during the combination of dopamine and dobutamine (each at 20 µg/kg/min i.v.). Following completion of the above protocol, the LAD was totally occluded and Mb- spectra were obtained.

2.9 Tissue preparation
In three animals with LVH, myocardial biopsies (approximately 10 mg each) were obtained with a biopsy forceps precooled to –70°C for determination of ATP and creatine content using an HPLC technique [11]. The heart was then fixed in 10% buffered formalin. The atria, right ventricle, aorta and large epicardial vessels were dissected from the LV. The LV was then sectioned into four transverse rings parallel to the mitral valve annulus so that a myocardial ring approximately 2.0 cm in thickness contained the region of myocardium located directly beneath the surface coil. The myocardium beneath the surface coil was removed and sectioned into three transmural layers from epicardium to endocardium, weighed and placed into vials for counting of radioactivity. Similar myocardial specimens were obtained from the lateral and posterior LV wall to insure that the measurements from the region beneath the surface coil were representative of the entire LV.

2.10 Determination of blood flow
Radioactivity in the myocardial and blood reference specimens was determined using a gamma spectrometer (model 5912, Packard Instrument Company, Downers Grove, IL, USA) at window settings corresponding to the radioisotopes used during the study. Activity in each energy window was corrected for overlap between isotopes as well as for background activity. Knowing the rate of withdrawal of the reference blood specimen (Q_r) and the radioactivity of the reference specimen (C_r), myocardial radioactivity (C_m) was used to compute myocardial blood flow (Q_m) as: Q_m=Q_rx(C_m/C_r). Blood flow was expressed as ml/min/g of myocardium.

2.11 Data analysis
Hemodynamic data were measured from the chart recordings. PCr and ATP during each experimental condition were expressed as a percent of the baseline value for each compound. ³¹P-NMR spectra from the first, third and fifth voxels were taken to represent subepicardium, midmyocardium and subendocardium, respectively. Hemodynamic, biochemical and blood flow data were analyzed with one-way analysis of variance with replications. A value of p<0.05 was required for significance. When the analysis of variance yielded a significant result individual comparisons were made using the method of Scheffé. Data are reported as mean±SEM.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
3.1 Anatomic data
Anatomic data are summarized in Table 1. In the normal animals LV weight/body weight ratios ranged from 4.27 to 5.98 g/kg while in animals with aortic banding LV weight/body weight ratios ranged from 7.05 to 15.8 g/kg and averaged 101% greater than normal (p<0.01).

View this table: [in this window] [in a new window]   Table 1 Anatomic data

 
3.2 Hemodynamic data
Hemodynamic measurements are shown in Table 2. Under basal conditions there was no significant difference in heart rate between normal animals and animals with hypertrophy. Aortic pressure distal to the constricting band was lower in LVH than in normal animals (p<0.05). LV systolic and end-diastolic pressures were significantly higher in LVH than in normal hearts (p<0.05) and the heart ratexLV systolic pressure product (RPP) was also significantly higher in the LVH group (p<0.05). In response to stepwise increases of catecholamine infusion, RPP increased significantly in both groups (p<0.05). Normal hearts showed greater increases of heart rate, whereas the hearts with LVH showed greater increases of LV systolic pressure during catecholamine infusion. Rooke and Feigl [12] reported that when using RPP, proportional changes in heart rate or systolic pressure had similar effects on MVO₂. The percent increase in RPP during the highest dose of catecholamine (dobutamine plus dopamine, 20 µg/kg/min each) relative to the baseline value was similar in normal (280±38%) and LVH animals (254±30%).

View this table: [in this window] [in a new window]   Table 2 Hemodynamic data from eight normal dogs and nine dogs with LVH

 
3.3 Myocardial blood flow and MVO₂
Under basal conditions mean blood flow per g of myocardium and the ENDO/EPI ratio (see Section 3.4) were not significantly different in the two groups (Table 3). Dobutamine produced dose related increases of myocardial blood flow which were transmurally uniform and similar between normal and LVH hearts. However, the combination of dobutamine plus dopamine caused a significantly greater increase in mean blood flow in hypertrophied than in normal hearts (Table 3). The ENDO/EPI ratio was not significantly different from unity during any experimental condition in either normal or LVH hearts.

View this table: [in this window] [in a new window]   Table 3 Myocardial blood flow data from eight normal dogs and nine dogs with LVH

 
MVO₂ data from a subset of six normal animals and six animals with LVH are shown in Table 4. Coronary arteriovenous oxygen extraction was not different between normal and LVH hearts during baseline conditions. Oxygen extraction did not change during catecholamine infusion in the normal hearts. Myocardial oxygen extraction tended to decrease in the hypertrophied hearts during combined infusion of dobutamine plus dopamine but this did not achieve statistical significance. Oxygen consumption per g of myocardium was similar in normal and hypertrophied hearts during baseline conditions and increased similarly in both groups during catecholamine infusion. Both normal and hypertrophied hearts demonstrated net lactate extraction during basal conditions; lactate extraction tended to decrease with increasing levels of catecholamine infusion, although this was not significant. Coronary venous PO₂ was similar in normal and hypertrophied hearts during baseline conditions and did not change significantly in either group during catecholamine infusion.

View this table: [in this window] [in a new window]   Table 4 Myocardial oxygen consumption and net lactate uptake data from six normal dogs and six dogs with LVH

 
3.4 ³¹P-NMR measurements
Myocardial PCr/ATP ratios and the changes during catecholamine infusions are summarized in Table 5. The voxel labeled EPI was located over the outer edge of the LV wall while the voxel most distant from the coil, labeled ENDO, was located over the subendocardium. The voxel labeled MID was located over the midmyocardium. During baseline conditions the PCr/ATP ratio in hypertrophied hearts was significantly lower than normal in each transmural layer (each p<0.05). There was a transmural gradient of PCr/ATP in both normal and hypertrophied hearts, with values in the ENDO voxel significantly less than in the EPI voxel (p<0.05). In response to dobutamine, 15 µg/kg/min, there was no significant change in PCr/ATP in either normal or hypertrophied hearts. When the dose of dobutamine was increased to 30 µg/kg/min, the PCr/ATP ratio tended to decrease in normal hearts, and this achieved statistical significance in the EPI and ENDO voxels. In the hypertrophied hearts this dose of dobutamine caused a significant decrease in the mean PCr/ATP for the whole LV wall as well as for each of the transmural voxels (each p<0.05). The decrease in PCr/ATP was significantly greater in hypertrophied than in normal hearts for all three voxels as well as for the whole LV wall (each p<0.05). During infusion of dobutamine plus dopamine (each 20 µg/kg/min) PCr/ATP decreased significantly only in the EPI voxel of the normal hearts. In contrast, PCr/ATP decreased significantly in all three voxels of the hypertrophied hearts. In the hypertrophied hearts the decrease of PCr/ATP was significantly greater than normal in the MID and ENDO voxels (p<0.05). A typical example of ³¹P-NMR spectra from the full thickness of the LV wall from a heart with LVH under baseline conditions, during infusion of dopamine plus dobutamine (each 20 µm/kg/min) and during coronary occlusion is shown in Fig. 1.

View this table: [in this window] [in a new window]   Table 5 PCr/ATP and Pi/PCr in eight normal dogs and nine dogs with LVH

 

View larger version (12K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Representative 31P-NMR spectra from an animal with left ventricular hypertrophy. Data are shown during (A) basal conditions, (B) infusion of dobutamine (20 µg/kg/min i.v.) combined with dopamine (20 µg/kg/min), and (C) coronary artery occlusion. PCr=phosphocreatine; Pi=inorganic phosphate. The three resonances (, , and β) of ATP are shown. Pi was not detected during basal conditions, but appeared both during catecholamine infusion and during coronary occlusion.

 
Myocardial Pi/PCr are shown in Table 5. During baseline conditions Pi was too low to identify at the signal-to-noise ratio (S/N) of the spectra. During dobutamine, 15 µg/kg/min, Pi could not be detected in any of the normal hearts but was detectable in five of the nine hypertrophied hearts. When the dose of dobutamine was increased to 30 µg/kg/min, Pi was detected in all transmural layers of both normal and hypertrophied hearts; Pi/PCr tended to be greater in hypertrophied than in normal hearts, and this achieved statistical significance in the EPI voxel. Infusion of dobutamine plus dopamine did not cause a further increase in Pi/PCr in the normal hearts, but tended to further increase the Pi/PCr in the hypertrophied hearts. During dobutamine plus dopamine Pi/PCr was significantly greater than normal in the EPI and MID voxels of the hypertrophied hearts.

Myocardial ATP content in three hearts with LVH was 17.4±0.5 µmol/g dry weight, 28% less than in normal historical controls in which a value of 23.9±0.8 µmol/g dry weight was obtained [8]. Myocardial creatine content for these three hearts with LVH was 100±3.2 µmol/g dry weight, 14% less than in normal historical canine hearts in which a value of 116±3 µmol/g dry weight was obtained [8].

3.5 ¹H Mb- measurements
Typical ¹H-NMR spectra from a normal heart and a heart with LVH are shown in Figs. 2 and 3, respectively. No Mb- was detected under basal conditions or during dobutamine or combined dobutamine plus dopamine infusion in either the normal group or the LVH group. During LAD occlusion a prominent Mb- resonance was observed at approximately 71 ppm down field from the water resonance in each of the hearts studied.

View larger version (6K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 1H-NMR spectra from a normal heart during baseline conditions (A), during infusion of dobutamine combined with dopamine (each 20 µg/kg/min i.v.) (B), and during total coronary occlusion (C).

 

View larger version (7K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 1H-NMR spectra from a heart with LVH during baseline conditions (A), during infusion of dobutamine combined with dopamine (each 20 µg/kg/min i.v.) (B), and during total coronary occlusion (C).

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
Several new findings are reported in this study. First, the abnormally decreased PCr/ATP ratio found in severely hypertrophied myocardium during basal conditions cannot be ascribed to inadequate myocyte oxygen availability. Second, the loss of PCr and accumulation of Pi which occur during high workloads produced by catecholamine infusion were not associated with myoglobin deoxygenation, indicating that these changes are not the result of oxygen insufficiency. Finally, the more prominent HEP changes during catecholamine stimulation in hypertrophied hearts were not associated with detectable Mb-, thus failing to support impaired oxygen diffusion as a limiting factor in the hypertrophied myocardium. The implications of these findings will be discussed in detail.

4.1 Myocyte oxygenation measurements
Knowledge of the level of intracellular oxygenation is needed to determine whether insufficient oxygen availability is acting to limit the rate or alter the kinetics of myocardial ATP synthesis. Myoglobin can serve as an indicator of the intracellular oxygen level. Myoglobin oxygen desaturation can be detected with ¹H-NMR spectroscopy; the deoxygenated Fe(II) state causes a hyperfine contact shift of the N proton signal of the proximal histidine which resonates at 79 ppm at 25°C [4]. Jue and associates [5, 6] demonstrated that this N proton resonance can be used to assess the degree of myoglobin oxygen desaturation in isolated rat hearts perfused with hemoglobin-free perfusate. We have recently adapted this technique for use in the in vivo canine heart [7]. We observed that under basal workstate conditions no Mb- resonance was detected. However, when a hydraulic occluder was applied to create a coronary artery stenosis sufficient to cause decreases of PCr/ATP and increases of Pi/PCr, a Mb- resonance appeared which was linearly related to the decrease of blood flow [7]. These previous data demonstrate that the method can detect myoglobin desaturation in the in vivo heart.

Failure to detect Mb- at baseline or during dobutamine stimulation in any heart, despite significant decreases of PCr/ATP and increases of Pi/PCr was not likely the result of inadequate sensitivity of the technique. The sensitivity of the ¹H-NMR method for detecting myoglobin desaturation would be expected to be high because of the high S/N ratio of the technique (Figs. 2 and 3), and because Mb- NMR visibility in muscle appears to be near 100% [13, 14]. This is in agreement with the finding that Mb has a rotational diffusion correlation time only slightly higher in the cytosol than in solution and does not appear to be compartmentalized within the myocyte [6]. These considerations suggest that Mb- should be detectable when myoglobin desaturation is greater than 10%. The range of reported values at which Mb is 50% saturated with oxygen (P₅₀) at physiological temperatures is 2.5–5 mmHg [15–17]. If a P₅₀ value of 2.5 mmHg is assumed at 37°C, a myoglobin oxygen saturation of 90% corresponds to an intracellular PO₂ >20 mmHg [17], a value far above the K_M value for O₂ with regard to cytochrome oxidase which is <1 mmHg [18]. Using isolated rat hearts perfused at 25°C with progressively lower perfusate oxygen tensions, Kreutzer and Jue [19] reported that the critical intracellular PO₂ for PCr is 2 mmHg. At oxygen tensions above this value PCr remained stable while below this value PCr decreased in proportion to the reduction of PO₂. In the present study PCr in the hypertrophied hearts fell to 78% of baseline during infusion of dobutamine plus dopamine. According to the data of Kreutzer and Jue [19] loss of PCr of this degree secondary to oxygen insufficiency would occur at an intracellular PO₂ of 1.8 mmHg. At a PO₂ of 1.8 mmHg more than 50% of the myoglobin would be in the deoxygenated state. Considering the robust resonance peak observed during total occlusion (corresponding to nearly 100% Mb-), 50% myoglobin desaturation would have been readily detected. Failure to detect Mb- implies that mitochondrial oxygen availability was nonlimiting and is incompatible with the concept that oxygen insufficiency was responsible for the loss of PCr observed during catecholamine infusion.

The affinity of Mb for oxygen is approximately an order of magnitude greater than that of Hb. This difference in affinity would allow Mb to facilitate oxygen diffusion from the capillaries into the myocyte by lowering the concentration of free oxygen at the sarcolemma [15, 16]. Furthermore, Mb can facilitate a large total oxygen flux while maintaining a shallow gradient of free oxygen from the sarcolemma to the interior of the cell. Microspectrophotometric studies have demonstrated that myoglobin saturation in flash frozen myocardial sections is homogeneous within a myocyte [16] so that measurements of average myocardial myoglobin saturation should reflect perimitochondrial PO₂. The present data indicate that myoglobin desaturation is not required to facilitate oxygen conductance in the myocardium even at the moderately high MVO₂ values achieved in the present study (which are approximately 50% of those during heavy exercise) [20, 21]. It may be that some degree of myoglobin desaturation would be required to facilitate oxygen conductance at the higher myocardial workstates achievable during intense exercise.

4.2 Technical considerations regarding Mb- measurements
Mb- and deoxyhemoglobin (Hb-) resonances partially overlap in skeletal muscle ¹H-NMR spectra [14, 22], but we believe that the resonance appearing during myocardial ischemia was primarily that of Mb- for the following reasons. During basal conditions in open chest dogs the heart extracts 50 to 70% of the oxygen from the coronary blood so that a substantial fraction of Hb in the heart is normally in the deoxygenated state. Myocardial Hb content has been reported to be 2.27 g/100 g of tissue [23] which corresponds to 0.355 µmole of Hb per g of myocardium. Since 50% of myocardial Hb is venous, and 50 to 70% of the venous Hb is in the deoxygenated form (Hb-), there must exist 0.09–0.12 µmoles of Hb-/g of heart tissue under basal conditions. However, we did not detect Hb- resonances either under basal conditions or during total coronary occlusion. This is consistent with previous reports of red blood cells studied in vitro. NMR visibility of Hb- in solution is high (and comparable to that of Mb-), but in intact red blood cells Hb- visibility was only 16% of the value observed in solution [22]. Increasing intracellular water by red cell swelling markedly increased the Hb- resonance [22], suggesting that the intracellular environment in normal red cells restricted Hb mobility, thereby decreasing NMR visibility. The limited Hb- visibility in vivo is further supported by studies in which an arterial occlusion was used to cause desaturation of both hemoglobin and myoglobin in the human forearm [13]. Although both Mb- and Hb- resonances were resolved, the Hb- resonance area was only about 10% that of Mb- [13, 22]. Failure to resolve Hb- resonances in the heart despite the low oxygen content of venous blood indicates that MR visibility of Hb- in cardiac muscle is also low. This is further supported by the finding that Mb- resonances were also not detected during total coronary occlusion.

4.3 HEP abnormalities in LVH
In a subset of three of the animals with LVH, chemically determined ATP was 28% less and PCr was 47% less than in historical control normal canine hearts studied in this laboratory during basal conditions [8]. We previously reported similar reductions of HEP content in this experimental model of LVH [8]. The decreased PCr could not be accounted for by a decreased creatine content, since total myocardial creatine was only 14% less in hypertrophied than in normal hearts. In a previous study of hypertrophied hearts infusion of adenosine to produce a threefold increase in coronary blood flow caused no change in MVO₂ and no increase in the PCr level or the PCr/ATP ratio, thus demonstrating that the decreased PCr was not the result of persistent underperfusion [8]. In the present study the decreased PCr/ATP ratio during basal conditions in the hypertrophied hearts was not accompanied by a detectable Mb- resonance, thus demonstrating that oxygen limitation was not the basis for the lower PCr/ATP ratio. During the high workstates the PCr/ATP ratio fell significantly with the appearance of Pi in all transmural myocardial layers. These changes were greater in hearts with LVH than in normal hearts despite comparable relative increases in RPP and oxygen consumption rate. It should be noted that these changes were observed at relatively high workloads; we [34] and others [35] have previously reported no change in PCr or ATP during catecholamine stimulation in the canine heart at lower levels of work (i.e. RPPs <35 000 mmHgxbeat/min). The HEP changes during catecholamine infusion were not associated with detectable myoglobin desaturation, indicating that cytosolic oxygen concentration remained high during the period of markedly increased ATP synthesis. In the absence of oxygen limitation, the decreased PCr/ATP ratio might reflect altered kinetics of oxidative phosphorylation in the LVH hearts. The myocardial PCr/ATP ratio is related to the cytosolic free ADP concentration because creatine kinase catalyzes a near equilibrium reaction in which a phosphoryl group is transferred between PCr and ATP with no loss of free energy [24]. However, cytosolic [ADP] may not be strictly comparable to [ADP] in the locale of the adenine nucleotide translocase where regulatory control is exercised [25]. Rather, the relative impermeability of the outer mitochondrial membrane to ADP and the close coupling of mitochondrial creatine kinase to the translocase imply some degree of compartmentation of intracellular ADP [25]. Thus, mitochondrial creatine kinase can act to maintain high ADP levels in the region of the translocase while eliminating the requirement for comparably high cytosolic [ADP], thus resulting in a decrease of the apparent K_M of cytosolic ADP with regard to MVO₂ [25]. Nevertheless, it is likely the changes in cytosolic ADP reflect directionally similar changes in intramitochondrial ADP.

It is possible that differences in substrate preference between normal and hypertrophied hearts could contribute to differences in myocardial ADP and Pi levels between the two groups. This hypothesis is supported by observations in perfused rabbits hearts in which a switch in substrate from glucose to pyruvate resulted in increased intramitochondrial NADH levels [26]. Furthermore, infusion of pyruvate was found to increase myocardial PCr in the canine heart in vivo with no change in MVO₂ [27]. These findings can be interpreted in terms of a model in which oxidative phosphorylation is kinetically regulated by its primary substrates, ADP, Pi, intramitochondrial NADH and O₂ [28]. Thus, a switch in substrate from pyruvate to glucose results in a decrease in the intramitochondrial NADH level; as a result cytosolic ADP and Pi levels are required to increase for any given rate of ATP synthesis. Previous data have demonstrated that glucose uptake is increased in hearts with LVH [29]; as in the isolated perfused heart studies, an increase in glucose utilization could contribute to higher ADP concentrations in the hypertrophied hearts [26, 28]. It is also possible that a change in activity of the creatine kinase system of the hypertrophied hearts could alter ADP levels. Myocardial hypertrophy is associated with a fetal shift in creatine kinase expression; a change in cytosolic creatine kinase activity or in mitochondrial creatine kinase content could influence ADP levels in LVH [30]. Alterations of the adenine nucleotide translocase might require higher ADP levels to support any level of oxygen consumption. Activity of the translocase is decreased in myocardium from patients and experimental animals with myocarditis and overt heart failure [31, 32], but no studies of the translocase have been reported for hearts with compensated hypertrophy. A final consideration is that although MVO₂ reflects the rate of ATP synthesis, a significant proportion of oxygen is consumed as a consequence of the mitochondrial proton leak [33]. It is unknown whether myocardial hypertrophy could increase the degree of proton leak, thereby altering the relationship between oxygen consumption and the rate of ATP synthesis.

	5 Conclusions

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 
Changes in HEP levels during basal conditions in severely hypertrophied myocardium are not caused by insufficient oxygen availability to the myocyte. Furthermore, decreases of PCr/ATP and appearance of Pi during high workstates in both normal and hypertrophied hearts cannot be ascribed to inadequate oxygen delivery. It is likely that the greater loss of PCr during high workstates in the hypertrophied hearts is the result of alterations in the regulation of myocardial oxidative phosphorylation. This HEP alteration did not restrict cardiac function during the increases in workload produced by catecholamine stimulation in the present study.

Time for primary review 32 days.

	Acknowledgements

 
This work was supported by US Public Health Service Grants HL21872, HL33600 and HL50470, Department of Veteran’s Affairs Medical Research Funds, and a Grant-in-Aid from the American Heart Association—national. Dr. Zhang is recipient of an Established Investigator Award from the American Heart Association.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusions References

 

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