INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

IN THE HEART, ATP is synthesized mainly in the mitochondria through oxidative phosphorylation and transported to the contractile apparatus, where it is consumed by myosin ATPase to generate force. The creatine kinase (CK) system plays an important role in myocardial energy metabolism by maintaining ADP levels high at the mitochondria, where ATP is generated, and low at sites of ATP utilization (21, 45). The latter is postulated to contribute to the maintenance of a high free energy of ATP hydrolysis, thereby enhancing the efficiency of the energy utilization processes (43). In addition, a CK shuttle has been proposed, wherein high-energy phosphate transport within the cell is facilitated by the higher diffusibility of creatine and phosphocreatine (PCr) relative to ADP (5, 44).

In postinfarction-remodeled hearts and in failing hearts, a fetal shift of myocardial CK expression has been reported, with a decrease in the MM-isoform and increases in the MB- and BB-isoforms (9, 14, 16-19). Similar findings were observed in a canine model of left ventricular (LV) hypertrophy (LVH) produced by ascending aortic banding (42). Although the mechanism and functional consequences of these CK isoform shifts in the overloaded and failing heart are unclear, previous studies have demonstrated that decreases of CK activity have the potential to affect myocardial performance. Thus in rats in which the CK system was inhibited with iodoacetate or by chronic feeding of a creatine analog, the ability of the heart to respond to an increased workload was impaired (22, 40). In transgenic mice lacking CK-MM and mitochondrial CK (CK-mito), a higher myocardial free ADP concentration was required to maintain ATP synthesis (33, 34). Consequently, the present study was performed to determine whether abnormal CK isoform expression during pressure-overload hypertrophy induces alterations in high-energy phosphate kinetics that limit contractile performance. mRNA and protein levels of the CK isoforms were examined in hypertrophied and normal hearts, and ³¹P magnetic resonance spectroscopy (MRS) was used to determine whether the change in isoform expression in the hypertrophied heart was associated with a change of the CK flux rate.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Studies were performed in accordance with the "Position of the American Heart Association on Research Animal Use," and protocols were approved by the Animal Care Committee of the University of Minnesota.

Production of LVH. Fourteen mongrel dogs (8 wk of age) were anesthetized with pentobarbital sodium (25-30 mg/kg iv), intubated, and ventilated with a respirator. A right thoracotomy was performed in the third intercostal space, and the ascending aorta, ~1.5 cm above the aortic valve, was mobilized and encircled with a polyethylene band 2.5 mm in width (2). When the LV and distal aortic pressures were measured, the band was tightened until a peak systolic pressure gradient of 20-30 mmHg was achieved across the narrowing. The chest was then closed, the pneumothorax was evacuated, and the animals were allowed to recover. LVH occurred progressively as the area of aortic constriction remained fixed in the face of normal body growth. At ~1 yr of age, animals were returned to the laboratory for study.

Experimental preparation. Fourteen animals with LVH, as well as ten normal animals that served as a control group, were premedicated with morphine sulfate (1 mg/kg sc) and anesthetized with -chloralose (100 mg/kg iv followed by an infusion of 10 mg · kg¹ · h¹). Animals were intubated and ventilated with a respirator with supplemental oxygen; arterial blood gases and pH were maintained within the physiological range. A polyvinyl chloride catheter (outer diameter, 3.0 mm) 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 was 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. An NMR surface coil was sutured to the anterior LV wall overlying the region perfused by the left anterior descending coronary artery. The surface coil was constructed of a single turn of copper wire and incorporated a 33-pF capacitor; surface coils were 28 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 was allowed to assume its normal position. Animals were placed on a circulating water heating blanket, which maintained body core temperature at 37 ± 1°C, and positioned within the magnet.

Myocardial blood flow. Myocardial blood flow was measured with microspheres (diameter, 15 µM) labeled with ¹⁴¹Ce, ⁵¹Cr, ⁹⁵Nb, ⁸⁵Sr, or ⁴⁶Sc (NEN; Boston, MA). For each measurement, ~3 × 10⁶ microspheres were administered into the left atrial catheter. A reference sample of arterial blood was withdrawn from the aortic catheter at a rate of 15 ml/min beginning 5 s before the microsphere injection and continuing for 120 s. Radioactivity in the myocardial and blood reference specimens was determined using a -spectrometer (Packard; Downers Grove, IL) at window settings chosen for the combination of radioisotopes used during the study. Activity in each energy window was corrected for overlapping activity and for background activity. As the rate of withdrawal of the reference blood specimen (Q_r) and the radioactivity of the reference specimen (C_r) was known, myocardial radioactivity (C_m) could be used to compute blood flow (Q_m) as follows: Q_m = Q_r × (C_m/C_r).

NMR technique. Measurements were performed in a 40-cm bore 4.7-T magnet interfaced with a Spectroscopy Imaging Systems (Fremont, CA) computer console as previously described (13, 31). The LV pressure signal was used to gate NMR data acquisition to the cardiac cycle, whereas respiratory gating was achieved by triggering the ventilator to the cardiac cycle between data acquisitions. Spectra were recorded in late diastole with a pulse repetition time of 6-7 s. This repetition time allowed full relaxation for ATP and P_i resonances and ~95% relaxation for the PCr resonances (31). PCr resonance intensities were corrected for this saturation. Radiofrequency (RF) transmission and signal detection were performed with previously described surface coils (13, 31, 46). A capillary containing 15 µl of 3 M phosphonoacetic acid was placed at the coil center to serve as a reference. The proton signal from water 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 (31). With the use of the static magnetic field magnitude gradient and adiabatic inversion pulses, signal origin was restricted to a column coaxial with the surface coil (perpendicular to the LV wall); column dimensions were 23 × 23 mm in LVH hearts and 18 × 18 mm in normal hearts (46, 47). Within this column, the signal was further localized to five voxels across the LV wall from epicardium to endocardium using the RF magnetic field magnitude generated by the surface coil gradient (31). 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 previously reported (13, 31). Each set of 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 parts per million relative to 85% phosphoric acid. Intracellular pH was determined from the chemical shift of P_i relative to the PCr resonance peak (4). Because of off-resonance problems associated with the ATP resonance peak, the ATP resonance was integrated for determination of ATP content. No baseline correction was used. Calibration of the epicardial voxel ATP content was performed using chemically determined ATP in an epicardial biopsy obtained at the conclusion of study.

Calculation of myocardial free ADP and P_i. Myocardial free ADP levels were calculated from the CK equilibrium expression using an equilibrium constant (K_eq) of 1.66 × 10⁹ and cytosolic pH = 7.1 as follows: [ADP] = ([ATP] [Cr_free])/([PCr] [H⁺] K_eq). PCr and ATP values were obtained from the spectra calibrated with the biopsy-measured ATP levels. Free creatine (Cr_free) was calculated by subtracting the PCr values from the biopsy measurement of total creatine. As reported by Katz et al. (23), the myocardial P_i resonance partially overlaps one of the resonances of 2,3-diphosphoglycerate (2,3-DPG) in erythrocytes in LV cavitary blood. As a result, the baseline P_i resonance in the present study was too small to be reliably separated from the 2,3-DPG resonance and from background noise. Consequently, P_i values were calculated as the difference between the integral of the P_i region during baseline conditions and during each experimental condition and are reported as P_i.

CK kinetics measured with ³¹P MRS saturation transfer. A double-tuned (200 MHz for ¹H and 81 MHz for ³¹P) surface coil (diameter, 28 mm) was used for RF transmission and signal detection as previously described (27). A chemical shift selective (CHESS) pulse sequence was employed to saturate the ATP resonance (12); this sequence consisted of a 90° Sinc RF excitation pulse followed by a short half-sine gradient pulse in all three orthogonal axes to enhance the dephasing of transverse magnetization. This CHESS sequence was applied repetitively to ensure complete saturation of the ATP resonance. The repetition time for signal acquisition of 12 s provided fully relaxed ATP and PCr resonances. Control spectra were acquired with the saturation carrier frequency setting on the opposite side of the PCr resonance with a frequency difference identical to that between PCr and ATP. The relative change of the PCr resonance intensity between the saturated and control spectra is proportional to the forward rate constant (k_f) in the exchange reaction between PCr and ATP (6, 27). All spectra were recorded with a spectral width of 6,000 Hz.

Hemodynamic measurements. Aortic and LV pressures were measured with fluid-filled pressure transducers at midchest level. Data were recorded on an eight-channel direct writing recorder (Coulbourne Instruments; Lehigh Valley, PA). Hemodynamic data were recorded continuously throughout the study. Arterial blood gases were measured every 15 min, and the respirator was adjusted to maintain PO₂, PCO₂, and pH in the physiological range.

Experimental protocol. Hemodynamic measurements and ³¹P MRS spectra were first obtained under basal conditions. Midway through the 10-min MRS acquisition period, a microsphere injection was performed for determination of myocardial blood flow. After baseline data acquisition was completed, the response to inotropic stimulation with dobutamine (15 µg · kg¹ · min¹ iv) was examined. After allowing 10 min to achieve steady-state conditions, we repeated all measurements in the ensuing 30 min. The dobutamine infusion rate was then increased to 30 µg · kg¹ · min¹ iv, and all measurements were again obtained.

Tissue preparation. At the conclusion of the study, an epicardial biopsy was taken from four normal and four LVH ventricles using a biopsy forceps precooled to 70°C for subsequent analysis of ATP content using HPLC (36). The animal was then euthanized, and the heart was excised. In six animals from each group, full-thickness myocardial specimens (~1 g each) were rapidly excised and frozen in liquid nitrogen for determination of CK isoform expression. Another full-thickness myocardial specimen (~3 g in weight) was frozen for determination of total creatine content (36). The heart was then fixed in 10% buffered formalin, and the LV myocardium beneath the surface coil was sectioned into three transmural layers from epicardium to endocardium, weighed, and placed into vials for counting of radioactivity.

Northern blot analysis for mRNA. RNA was extracted from frozen tissue samples (50-100 mg) using a commercial procedure (7). Cell components were disrupted and separated by centrifugation. RNA in the aqueous phase was precipitated with isopropanol and centrifuged, and the pellet was air-dried and reconstituted in RNase-free water. RNA (20 µg) was size fractionated by electrophoresis on 1% agarose formaldehyde gel in MOPS buffer for 3 h at 110 V; RNA standards (GIBCO-BRL; Grand Island, NY) were run with each gel. The RNA was then transferred to a Nytran (nylon) membrane using HETS transfer solution (Tel-Test; Friendswood, TX), prehybridized in denatured salmon sperm DNA in a Techne hybridizer (HB-1D; Cambridge, UK), and reacted with specific radioactive probes. A 100-bp human cDNA probe specific to CK-B (14), a 1,000-bp rat cDNA probe specific to CK-M (14), and a complementary DNA probe specific to the CK-mito subunit mRNA (14) were used. A Prime-It II random primer labeling kit (Stratagene) was used to radiolabel the probes with ³¹P-labeled nucleotides. The membrane was washed and placed in a plastic bag for autoradiography. The hybridization signal was quantitated using PhosphoImager SI (Molecular Dynamics). The membrane was stripped and sequentially reprobed after repeat prehybridization.

Protein extraction and Western blot analysis. Frozen heart samples (~50 mg) were added to 1 ml of ice-cold buffer [0.2 M potassium phosphate (pH 7.4) containing 5 mM EGTA, 5 mM -mercaptoethanol, and 10% (vol/vol) glycerol] to release mitochondrial and cytoplasmic enzymes. Samples were homogenized at 4°C for 20 s, followed by incubation with gentle agitation and centrifugation, such that the supernatant contained the isoenzymes in an optimal yield (32). Protein concentration was determined using a modified Lowry method (Sigma Diagnostics). Myocardial supernatants were electrophoresed on a commercial agarose gel system (Corning ACI, CIBA Corning Diagnostic; Medfield, MA). Commercially prepared molecular-weight standards and purified proteins (CK-MM, CK-MB, CK-BB, and CK-mito; all obtained from Aalto) were run as controls. The protein subunits were transferred for 1 h at 100 V in transfer buffer (25 mM Tris, 192 mM glycine, and 20% methanol). Monoclonal mouse antibodies specific to CK-M and CK-B (OEM Concepts; Toms River, NJ) were sequentially directed against their respective protein subunits bound to the membrane. The membrane was incubated with the appropriate secondary horseradish peroxidase-labeled (anti-mouse) IgG antibody, washed in Tween-Tris-buffered saline solution before a 1-min incubation with enhanced chemiluminescent substrate (ECL, Amersham), and exposed to X-ray film (XAR-5, Eastman Kodak) for 15 s-10 min. Densitometry was used for relative quantitation of the CK protein subunits.

Total CK and CK activity. Myocardial homogenates were prepared in ice-cold phosphate buffer [0.2 M potassium phosphate (pH 7.4) containing 5 mmol/l EGTA, 5 mmol/l -mercaptol ethenyl, and 10% glycerol] to release cytoplasmic and mitochondrial enzymes. Total CK activity in the supernatant was measured at 37°C in a centrifugal spectrophotometric analyzer (Cobas Bio, Roche Analytical Instruments; Nutley, NJ) using N-acetylcysteine (NAC)-activated reagents (reagent for CK NAC, Roche Analytical Instruments) (32). Enzyme results were expressed as international units of activity per milligram of total protein. Duplicate samples were electrophoresed and incubated with and without substrate (PCr) to exclude non-CK artifacts.

Data analysis. Hemodynamic data were measured from the chart recordings. Numerical values for PCr and ATP during each experimental condition were expressed as a percentage of the baseline value. ³¹P NMR spectra from the first, third, and fifth transmural voxels were taken to represent the subepicardium (Epi), midmyocardium, and subendocardium (Endo), respectively (31). 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 ANOVA yielded a significant result, individual comparisons were made using the method of Scheffé's. Data are reported as means ± SE.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Anatomic data. In 10 normal control animals, body weights ranged from 17.5 to 22.0 kg (mean = 20.0 ± 0.5 kg), LV weights ranged from 82.8 to 113.6 g (mean = 94.7 ± 3.9 g), and LV-to-body weight ratios ranged from 3.99 to 5.98 g/kg (mean = 4.91 ± 0.26 g/kg). In animals with aortic banding, body weights ranged from 17.5 to 30.0 kg (mean = 21.7 ± 1.2 kg) and LV weights ranged from 154.3 to 299.3 g (mean = 201.8 ± 13.7 g) and were significantly greater than normal (P < 0.01). LV-to-body weight ratios in animals with aortic banding ranged from 6.75 to 15.8 g/kg (mean = 9.45 ± 0.63 g/kg) and averaged 92% greater than in the normal animals (P < 0.01). None of the animals with LVH had clinical evidence of heart failure.

Hemodynamic data. Hemodynamic measurements are shown in Table 1. Under basal conditions, heart rates and mean aortic pressures were similar in normal and LVH animals. LV systolic and end-diastolic pressures were higher in LVH than in normal hearts (P < 0.05), and the heart rate × LV systolic pressure product [rate-pressure product (RPP)] was also higher in the hypertrophy group (P < 0.05). In response to the first dose of dobutamine, the RPP increased significantly in both groups (Table 1; P < 0.05). Normal hearts showed a greater increase of heart rate, whereas LVH animals showed a greater increase of LV systolic pressure. During dobutamine (30 µg · kg¹ · min¹) treatment, both groups had further increases of heart rate, LV systolic pressure, and RPP. However, the increase of LV systolic pressure reached significance only in normal hearts (Table 1). Mean aortic pressure and LV end-diastolic pressure did not change significantly during catecholamine infusion in either group (Table 1).

Myocardial blood flow and oxygen consumption. Under basal conditions, blood flow per gram of myocardium was similar in the two groups (Table 2). The Endo-to-Epi flow ratio tended to be lower the hearts with LVH, but this difference was not significant. In response to the first dose of dobutamine, both groups showed increases of myocardial blood flow, but in the normal animals this was significant only in the subepicardial layer (Table 2). During dobutamine (30 µg · kg¹ · min¹), there was a further increase in myocardial blood flow. In the animals with LVH, this increase tended to be greater in the Epi layer, although the difference was not significant. The myocardial oxygen consumption (MO₂) measured in six normal hearts and six hearts with LVH is shown in Table 3. There was no significant difference in oxygen extraction or in oxygen consumption per gram of myocardium between normal and LVH hearts.

View this table: [in this window] [in a new window]   Table 3.   MO2 in normal hearts and hearts with LVH

³¹P NMR measurements. Transmural sets of ³¹P NMR spectra from a normal heart and from a heart with LVH under baseline conditions and during dobutamine infusion are shown in Fig. 1. Myocardial PCr/ATP and P_i/PCr are summarized in Table 4. Spectra recorded during the control period were characterized by high PCr and ATP levels in both the normal and LVH hearts, whereas P_i was too low to identify at the signal-to-noise ratio of the spectra. However, in response to dobutamine stimulation, both groups showed decreases of PCr/ATP and increases of P_i/PCr across the LV wall. These changes were most pronounced in the hearts with LVH (Fig. 1 and Table 4).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Transmurally differentiated 31P NMR spectra under basal conditions (A) and during dobutamine infusion (B) from a normal heart and a heart with left ventricular hypertrophy (LVH). Each transmural data set consists of a stack of 5 spectra corresponding to voxels from epicardium to endocardium. Endo, subendocardial voxel; Mid, midwall; Epi, subepicardial voxel. The phosphocreatine (PCr)-to-ATP ratios are decreased in LVH hearts, and this was more severe during dobutamine stimulation. *Spikes corresponding to Pi.

View this table: [in this window] [in a new window]   Table 4.   Myocardial PCr/ATP and Pi/PCr in normal dogs and dogs with LVH

Biopsy data. Compared with normal hearts (n = 4), ATP and total creatine contents were decreased by 34 and 15% in LVH hearts (n = 4, each P < 0.05; Table 5). The rate of ATP synthesis, calculated from the MO₂ values using the ratio P:O = 3 (assuming that mitochondrial uncoupling was not present), are also included in Table 5.

CK isoform expression. As shown in Table 6 and Figs. 2 and 3, in LVH hearts, CK-M mRNA was decreased by 40% (P = 0.05) and CK-M protein was decreased by 50% (P < 0.01). CK-B mRNA was increased, with the ratio of mRNA to 18S rRNA of 0.22 ± 0.09 compared with 0.0 ± 0.0 in the normal hearts (P < 0.01); CK-B protein was increased by more than 10-fold in the LVH hearts (P < 0.01). CK-mito protein was decreased by 22% (P < 0.05) in LVH hearts.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   A: Western blots of creatine kinase (CK) isoform proteins and -actin protein from 6 normal hearts and 6 hearts with LVH. B: densitometric intensities for protein bands from Western blots of CK-M, CK-B, and mitochondrial CK (CK-Mt) isoforms normalized to -actin. Values are means ± SE; n = 6 animals in each group. In LVH hearts, the protein levels of CK-M and CK-Mt isoforms were significantly decreased, CK-B was increased, and -actin was unchanged.

View larger version (41K): [in this window] [in a new window]   Fig. 3.   A: Northern blots of CK isoform mRNAs and 18S rRNA from 6 normal hearts and 6 hearts with LVH. B: densitometric intensities for mRNA bands from Northern blots of CK-M and CK-B isoforms normalized to 18 S rRNA. Values are means ± SE. In LVH hearts, the mRNA levels of CK-M were significantly decreased, whereas CK-B mRNA was significantly increased.

Total CK activity. Total CK activity was 228 ± 94 and 306 ± 76 IU · mg wet weight cardiac protein¹ · min¹ · g dry weight¹ for normal and LVH hearts, respectively (P = not significant).

CK kinetics. The CK kinetic data are summarized in Table 7. Under basal conditions, the M/M₀ ratio (which is linearly related to the CK flux rate) was similar in normal and LVH hearts. There was no significant change in M/M₀ in response to dobutamine in either normal or LVH hearts (Table 7 and Figs. 4 and 5). T₁ and k_f were not different between normal and LVH hearts.

View larger version (9K): [in this window] [in a new window]   Fig. 4.   31P NMR saturation transfer spectra from an LVH heart under basal conditions. A chemical shift selective (CHESS) pulse sequence consisting of a short hard pulse train was used to saturate the ATP resonance (arrow in B). The hard pulse width was 22-24 µs, and the interpulse delay was 0.53 ms. This CHESS pulse train provided a frequency-selective saturation with a narrow band. A 6-s saturation pulse train was used to achieve steady saturation. The repetition time (TR) for acquisition was 12 s, which was trigged according the respiration and cardiac cycle. This TR provides full-relaxation ATP and PCr resonances. The control spectra (A) was acquired with the saturation pulse frequency setting on the opposite side of the PCr resonance with a frequency difference identical to that between PCr and ATP. The relative change of the PCr resonance intensity between the saturated (B) and control spectra (A) was proportional to the forward rate constant of CK. ppm, Parts per million.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Stacked plots of 31P NMR spectra from a progressive saturation transfer experiment of a heart with LVH. The repetition time was 12 s. Arrow, frequency of ATP, which is saturated. The saturation time of the 7 spectra (y-axis) increases as follows: 0.1, 0.2, 0.4, 0.8, 1.6, 3.0, and 6.0 s, respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The hearts with LVH in the present study were characterized by significant decreases of myocardial PCr, creatine, and the PCr-to-ATP ratio as well as a 30% reduction of ATP. In animals with pacing-induced heart failure, Shen et al. (38) observed that myocardial creatine fell progressively as heart failure developed so that the PCr-to-ATP ratio remained essentially unchanged. In the present study, myocardial total creatine content was decreased in the hypertrophied hearts, but this decrease was insufficient to maintain a normal PCr-to-ATP ratio, implying that myocardial free ADP was increased (Table 6). When ADP is increased, adenylate kinase (myokinase) is activated to catalyze the transfer of a phosphoryl group between two molecules of ADP to generate ATP and AMP (11, 21). The increased AMP by itself or in conjunction with allosteric stimulation of 5'-nucleotidase augments conversion of AMP to adenosine (25). Unlike the adenine nucleotides, which do not cross the sarcolemma, adenosine can cross the cell membrane to enter the interstitial space, where it is further degraded to inosine and hypoxanthine and carried out of the heart by the coronary circulation (15). Loss from the adenine nucleotide pool is of considerable consequence, because regeneration of ATP must then occur through de novo synthesis, a slow and energy-costly process (25). Cumulative loss of ATP during episodes of exercise or other stress exceeding the ability for de novo synthesis could explain the depletion of myocardial ATP observed in the hypertrophied ventricles.

Myocardial CK isoform expression. Previous studies of experimental models of compensated cardiac hypertrophy have reported an increase of the fetal isoenzymes CK-MB and CK-BB, generally without change of CK-MM or CK-mito and no change in total CK activity (18, 42), whereas reductions of total CK activity and CK-mito were reported to occur in the setting of cardiac failure (9, 16, 17, 19). These results suggested that the accumulation of B- containing CK isoenzyme is a marker of hypertrophy, whereas decreases of CK-MM and CK-mito are markers of pump failure. In the present study, Western blot analysis demonstrated a 50% decrease of CK-M protein in the hypertrophied hearts as well as a marked increase in CK-B protein. The results of Northern blot analysis with probes specific for CK-M and CK-B demonstrated that the change in protein isoenzyme expression was mediated at the level of mRNA abundance. Although posttranscriptional influences on mRNA stability cannot be excluded, previous studies in myogenic cell lines have demonstrated that the CK-M and CK-B genes are regulated at the level of transcription (41). In addition to the decreased CK-M protein, the animals with hypertrophy in the present study demonstrated a 22% decrease of CK-mito protein, but total CK activity per milligram protein was not significantly different between the hypertrophied and normal hearts. Although detailed evaluation of myocardial contractile performance could not be performed in the magnet during these studies, the animals demonstrated no evidence of cardiac failure. LV end-diastolic pressure was modestly increased, but this likely was the result of a decrease in chamber compliance secondary to the increased wall thickness. Thus the present study indicates that severe pressure-overload hypertrophy can be associated with significant reductions of both CK-mito and CK-M protein expression in the absence of overt LV failure.

Myocardial free ADP levels. Oxidative phosphorylation involves the synthesis of ATP from ADP and P_i, coupled to reduction of oxygen using electrons extracted from mitochondrial NADH. The relative importance of each substrate [ADP, O₂, P_i, and intramitochondrial NADH (NADH_m)] is determined by its level compared with its limiting Michaelis-Menten constant (K_m) and inhibition constant. Thus studies in perfused hearts have demonstrated that it is possible to achieve the same ATP synthesis rate at distinctly different intracellular levels of ADP, ATP, and P_i (10). For example, MO₂ (ATP synthesis rate) can be maintained despite a decrease of NADH_m, but only if ADP and/or P_i are appropriately increased (10). Possible explanations for the higher ADP levels in the hypertrophied hearts in the present study include decreased oxygen availability to the mitochondria; an increased K_m value for oxygen with regard to cytochrome aa₃; reduced P_i availability to the mitochondrial ATPase; reduced NADH generation resulting from either inadequate exogenous carbon substrate or disordered intermediary metabolism; altered function of the electron transport chain so that a lower mitochondrial proton gradient is maintained; or altered properties of the mitochondrial ATPase or the adenine nucleotide translocase so that the maximum velocity or K_m values with respect to ADP are increased (28). We (3, 48) have previously demonstrated that deoxymyoglobin is undetectable in normal or hypertrophied hearts during either basal conditions or catecholamine stimulated increases of work state, so that oxygen insufficiency cannot account for the increased ADP level. Although limitation of carbon substrate delivery to the myocyte is unlikely, alterations of substrate preference have been described in the hypertrophied heart, including decreased free fatty acid uptake and increased glucose uptake and glycolysis (1, 8, 46), with an increased ratio of inactive to active pyruvate dehydrogenase (37). These abnormalities could result in lower steady-state NADH_m levels and might also limit the rate of NADH_m generation (10). A possible consequence of these alterations would be a compensatory increase of cytosolic ADP to maintain ATP production at the level required by the ATP utilization rate. This alteration might explain, at least in part, the increased ADP levels in the hypertrophied hearts.

Myocardial CK flux rates. Because the CK activity measured in myocardial homogenates was not significantly different between normal and LVH hearts, one might expect that the higher ADP levels in the hypertrophied hearts would result in greater forward flux through the CK reaction. However, the mean CK activity determined in myocardial homogenates neglects the effects of the intracellular localization of the CK isoenzymes. Thus CK-MM located in association the myosin ATPase consumes ADP produced during contraction to regenerate ATP for use by the contractile apparatus (43). CK-mito localized in association with the adenine nucleotide translocator can utilize ATP to generate PCr, thereby maintaining high local ADP levels available for ATP synthesis but relatively lower mean cytosolic ADP levels (45). Because the CK-B isoform is not similarly localized within the myocyte, the increased CK-B in the hypertrophied heart may not reproduce the specific functions of CK-MM and CK-mito but does contribute to chemically determined total CK activity.