bioenergetic consequences of cardiac phosphocreatine depletion

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Val. 266, No. 30, Issue of October 25, pp. 2029620304,1991 Printed in U.S.A.

Bioenergetic Consequences of Cardiac Phosphocreatine Depletion Induced by Creatine Analogue Feeding*

(Received for publication, November 15, 1990, and in revised form, July 12, 1991)

Jay L. ZweierS and William E. JacobusSQll From the Peter Belfer Laboratory for Myocardial Research in the $Department of Medicine and the §Departments of Biological Chemistry and Anesthesiology and Critical Care Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

Borivoj Korecky and Yvonne Brandejs-Barry From the Department of Physiology, The University of Ottawa, Ontario KlH8M5, Canada

To further evaluate the bioenergetic role of phosphocreatine, we assessed several parameters in normal and depleted rat hearts. Rats were fed (8 weeks) a diet containing either 1% &guanidinoproprionic acid or 2% B-guanidinobutyric acid @-GBA), resulting in an 80% phosphocreatine depletion compared to controls. Left ventricular pressure-volume curves were obtained to determine contractile function. At any volume, the developed pressure in depleted hearts was lower than in controls. At the plateau, the rate-pressure product was between 37-45% lower: 34,000 @-GBA), 30,174 (8-guanidinoproprionic acid) versus 54,400 (control).

"P NMR spectroscopy on 8-GBA-treated hearts obtained the [ATP] and [phosphocreatine], which with saturation transfer estimated the rates of creatine kinase and ATP production. In depleted hearts, the rate constant for ATP synthesis from phosphocreatine was increased 33%. However, the flux was 72% lower. ATP production from ADP and Pi were similar under normal conditions, in spite of higher rates of oxygen consumption in the depleted hearts. The addition of 50 mM creatine to control perfusate had no effect on function or high energy phosphates. In contrast, a 28% increase in function and a 52% increase in [phosphocreatine] was seen in &GBA hearts. There was a marked increase in free [ADP] in B-GBA hearts, resulting in a lower estimated ATP phosphorylation potential. Over- all, the results suggest that phosphocreatine may play an important function by optimizing the thermody- namics of cardiac high energy phosphate utilization.

Over the past six decades the function of phosphocreatine (PCr)' and creatine kinase in cardiac energy metabolism has been extensively studied but is still not completely understood (3). At present, there are two conflicting hypotheses regarding the role of PCr in the heart. Shortly after its isolation, it was

* This work was supported by United State Public Health Service Grants HL-20658, HL-33592, and P50 HL-17655 for the Specialized Center for Ischemic Heart Disease and by the Heart and Stroke Foundation of Ontario. Preliminary reports of a portion of this work have been published (1,2).

Medical College of Ohio, P. 0. Box 10008, Toledo, OH 43699-0008. 1 To whom reprint requests should be addressed Dept. of Anatomy,

The abbreviations used are: PCr, phosphocreatine; Cr, creatine; LVDP, left ventricular developed pressure (systolic-diastolic pressure); @-GBA, @-guanidinobutyric acid @-GPA, P-guanidinoproprionic acid; HEPES, 4-(2- hydroxyethy1)-1-piperazineethanesulfonic acid EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid MDH, malate dehydrogenase.

recognized that the hydrolysis of phosphocreatine may pro- vide the driving energy for muscle contraction (4). Alterna- tively, the characterization of the myosin-ATPase reaction (5) led to the notion that ATP generated by metabolic proc- esses directly diffused to the myofibrils where it was utilized to fuel contraction (6). According to this latter view, PCr was a nonessential energy store or merely an energy reserve, with little or no direct functional importance. More recently it was demonstrated that there are both myofibrilar and mitochondrial isozymes of creatine kinase. Based on this fact, it was proposed that PCr functions as a shuttle molecule, thereby coupling mitochondrial ATP production to sarcoplasmic ATP utilization (7-1 1).

In view of the low cellular ADP concentrations of only approximately 40 p~ (12), the PCr shuttle is thought to play an important role in facilitating the recycling of a secondary phosphate acceptor, Le. creatine, back to the mitochondria to maintain the rates of oxidative phosphorylation (13). Hence ATP production remains in concert with contractile utilization (14). The existence of this PCr shuttle is thought to be of particular importance to maintain sustained high perform- ance levels (1, 2, 15-17). However, major controversy has again developed regarding the validity of the PCr shuttle hypothesis (18-21).

In order to probe in depth some of these questions, we depleted hearts of PCr by feeding the Cr analogues @-GPA and P-GBA. We then measured contractile function, O2 consumption rates, concentrations of high energy phosphates, and rates of creatine kinase and ATP synthesis in control and these depleted rat hearts. The results obtained clearly demonstrate fundamental biochemical, physiological, and thermodynamic differences between control and PCr-depleted hearts.

MATERIALS AND METHODS

Animal Preparation-Male Sprague-Dawley rats were fed a diet containing 2% P-GBA for 8 weeks. A paired matched fed group was also prepared. The weights of the rats were approximately 250-300 g at the start of feeding and 400 g after feeding. @-GBA was synthesized from @-amino butyric acid and cyanamide, according to the procedure of Rowley et al. (22). Depletion of creatine with 8-GPA, purchased from Aldrich, was similarly achieved by feeding a diet containing 1% P-guanidinoproprionic acid for 8 weeks.

Perfused Heart Preparation-Hearts were removed from the deeply anesthesitized animals and retrograde perfused in an isovolumic mode by a modification of the method of Langendorff, at a constant coronary arterial perfusion pressure of 80 mmHg. Heart rate and left ventricular-developed pressure (LVDP) were measured using a fluid- filled balloon secured into the left ventricle. The balloon was con- nected to a Statham P23dB pressure transducer via a hydraulic line and the transducer output amplified to a strip chart recorder so that

20296

Effects of Phosphocreatine Depletion on Heart Bioenergetics 20297

LVDP and its first derivative, dP/dt, could be simultaneously recorded. Hearts were paced using a 3 M KCl, 1% agar wick electrode secured in the right ventricle via the pulmonary artery. The Krebs- bicarbonate buffer contained 117 mM NaC1, 24.6 mM NaHC03, 5.8 mM KCl, 1.2 mM MgC12, 3.0 mM CaC12, 16.7 mM glucose, 1.1 mM mannitol, and was vigorously bubbled with 95% 0 2 , 5% COZ gas, resulting in a buffer pH of 7.4. There was no Pi in the perfusate. No adjustments in osmolarity were made when creatine (50 mM) was added directly to the perfusate.

Zsohtwn of Mitochondria-Mitochondria were isolated from rat hearts by a trypsin digestion method (23). Ventricular tissue from each heart was minced, washed, and suspended in 20 ml of isolation medium: 70 mM sucrose, 10 mM K-HEPES, pH 7.2,210 mM mannitol, and 0.5 mM EGTA. The tissue was subjected to mild trypsin digestion (2.5 mg) for 15 min at 0 'C, then diluted with 20 ml of isolation medium at pH 7.2 containing 1 mg/ml bovine serum albumin and 13 mg of soybean trypsin inhibitor. The suspension was stirred and the supernatant decanted. The partially digested tissue was then resuspended in 20 ml of isolation medium containing 1 mg/ml bovine serum albumin, homogenized briefly (10-15 s) with a loose-fitting Teflon-glass homogenizer, followed by complete disruption with a tight-fitting pestle (10 8) . The homogenate was centrifuged for 10 min at 600 X g (4 "C). The supernatant was decanted and centrifuged at 8000 X g for 10 min to obtain the mitochondrial fraction. The upper fluffy layer was discarded, and the tightly packed dark pellet was resuspended twice in 20 ml of isolation medium containing 1.0 mg/ml bovine serum albumin, each time followed by centrifugation for 10 min at 8000 X g (4 "C). The final washed pellet was suspended in isolation medium containing 1.0 mg/ml bovine serum albumin to a mitochondrial protein concentration of 50-100 mg/ml. Protein was determined by a biuret method (24), using crystalized, nitrogen- standardized, bovine serum albumin as the primary standard. Mito- chondrial preparations isolated by this procedure uniformly exhibited State 3 respiratory rates of 300-350 ng atoms of oxygen/min/mg of protein at 37 "C, and respiratory control values above 6 (6.0-10.0) in the presence of 1.0 mM M$+.

Oxygraph Procedures-Respiratory rates were assayed with a Yel- low Springs Clark electrode assembly in a closed oxygraph chamber, using a medium containing 0.13 M KC1, 10 mM K-HEPES, pH 7.2,5 mM glutamate, 5 mM malate, 1 mM M&12, 2 mM potasium phosphate, and 0.5 mM EGTA. The oxygen solubility in this medium was 400 ng atoms/ml at 37 "C (25). Cytochrome oxidase activity was measured polarographically as described by Schnaitman and Greenawalt (26).

Electron Microscopy-Sprague-Dawley rats (n = 4) of either the fed control, j3-GPA, or j3-GBA fed animals were anesthetized with Somnotol (50 mg/kg), heparinized (200 IU), and perfused throught the carotid artery with a fixative containing 0.045 M cacodylate buffer, 1.5% gluteraldehyde, 400 mOsmol/kg H20, maintained at pH 7.4. Upon fixation, the hearts were excised and cut into 2-mm pieces and fixed for 2 h in 2% osmic acid and 150 mM cacodylate buffer. They were then embedded in Araldite (520) for 12 h at 40 "C, followed by 24 h at 60 "C and then 18 h at 90 'C. Semi-thin and thin sections were cut with a diamond knife. The sections were stained for 30 min at 25 "C with uranyl acetate (saturated in 50% methanol, pH 4.4), and counter-stained for 20 min at 25 "C with Reynold's lead citrate, pH 12.0. Random samples of subendocardial, midwall, and subepicar- dial sections were selected for analysis and examined with a Philips Elu electron microscope.

31P Nuclear Magnetic Resonance-Pulsed Fourier transform NMR spectra were obtained using a modified Bruker WH-180 spectrometer interfaced to a Nicolet 1280 computer, with a 8.9- cm wide-bore 4.25- tesla superconducting magnet. A custom designed 20-mm 31P probe was tuned to the phosphorus resonance frequency of 72.88 MHz. The isolated perfused hearts were placed in 20-mm NMR tubes with all the requisite lines secured to an attached rod. All spectra were obtained using quadrature detection, a 60" flip angle, a sweep width of 3000 Hz, 4096 data points, and no proton decoupling. The interpulse delay was 2 s. Intracellular pH was calculated from the chemical shift, 80, of the Pi peak relative to the PCr peak, using the equation:

P H ~ = PK - loglo(& - G E / ~ A - 8,)

where pK = 6.90, 6, = 3.290 ppm, and 8E = 5.805 ppm as reported previously (27). Quantitation of the peak intensities of the spectra was performed either by on-line integration or by manual integration of the plotted spectra. Known concentrations of M$+ tri-metaphos- phate were placed in the ventricular balloon to serve as an internal absolute intensity standard for the determination of the tissue con-

centrations of the high energy phosphate metabolites (12). Saturation Transfer Experiments-These were performed using a

laboratory built 31P homonuclear decoupler with power output of 12 watts. The decoupler power was adjusted with an external attenuator to the minimum level required for complete resonance saturation. Control spectra were obtained with the decoupler frequency offset downfield from the PCr resonance by a frequency shift equal to that of the upfield offset from the y A T P resonance. Under these conditions, no decrease in the PCr resonance was observed. Thus there was no perturbation of adjacent signals by the selective saturating frequency. The experimental approach of performing the saturation transfer experiments was similar to that described previously (see Ref. 12, page 8017, for a more complete presentation of the theory and terms). The TU value in the absence of chemical exchange was measured from a plot of In (Mf - MA,) uersus t (12). With this value of TIA, the rate constant K was calculated from the equation:

K = l /TIA(M?/ML - 1)

Oxygen Consumption Measurements-Rates of heart oxygen consumption were determined by measuring the oxygen tension of the perfusate at the aortic cannula and the coronary effluent, sampled from a cannula placed through the pulmonary artery into the right ventricle. The inferior and superior venae cavae were ligated. Oxygen tension measurements were performed using a Radiometer Copen- hagen ABL2 blood gas analyzer. Myocardial oxygen consumption was calculated from the product of the measured coronary flow and the oxygen gradient (arterial-venous difference).

Measurement of Total Creatine and GBA from Acid Extracts- Hearts were frozen at 77°K with Wollenberger tongs, and the tissue was extracted in cold perchloric acid (12). Total creatine was measured by the method of Dubnoff (28) and j3-GBA by the Sakaguchi reaction, as described by Bonas et al. (29). Since j3-GBA gave some positive reaction with the creatine assay, appropriate corrections were performed for the falsely elevated creatine levels.

Enzyme Assays-The assay for creatine kinase was the spectro- photometric method of Eppenberger et al. (30), determined in the reverse direction. The rate of ATP formation was measured by a hexokinase/glucose-6-phosphate dehydrogenase method, and activity calculated from the increase in absorbance accompanying the formation of NADPH, recorded at 340 nm in a Gilford model 2400 spectrophotometer. The 3.0-ml assay medium contained 100 mM Tris- C1, pH 7.4, 0.15 mM NADP+, 3.3 mM MgCl,, 3.3 mM glucose, 0.5 mM ADP, 8.3 mM phosphocreatine, 5 pg of hexokinase (140 IU/mg), 5 pg of glucose-6-phosphate dehydrogenase (140 IU/mg), and 1.33 mM AMP to inhibit adenylate kinase. The assay was conducted at 30 "C with the reaction initiated by the addition of 12 pg of mitochondrial protein or 15 pg of homogenate protein. Malate dehydrogenase (MDH) activity was measured by the method of Ochoa (31).

RESULTS

Functional Characterization Measurements of Heart Function-Marked differences were

observed in the left ventricular function of the B-GBA, 8- GPA, and control hearts, Fig. 1. Measurement of LVDP was performed as a function of venticular balloon volume. These pressure volume measurements were performed on 14 control, 14 B-GPA hearts, and 14 8-GBA hearts. The curves obtained demonstrated that for any given LV volume both the 8-GPA and /3-GBA hearts exhibited much lower values of LVDP than in the controls (Fig. 1). At the plateau of the pressure-volume curve, the rate-pressure product was 37% lower in the 8-GBA hearts, 34,000 versus 54,400 in the controls, and 45% lower in the 8-GPA hearts, 30,174 (Table I). The balloon volume was fixed at the volume yielding the maximum LVDP and O2 consumption measurements were performed. Interestingly, in spite of the lower contractile function in the B-GBA or 8- GPA hearts, the oxygen consumption rates were actually slightly higher than in the controls, 8.7 or 8.9 versus 7.5 pmol/ g wet weightlmin.

Mitochondrial Studies-In order to determine if the observed functional depression in the P-GBA hearts was due to mitochondrial abnormalities or uncoupling, mitochondria

20298 Effects of Phosphocreatine Depletion on Heart Bioenergetics

250 r" A . . . . . . . . A . . . . . . . . A

.""."". A. . . . A CONTROL

0-0 GBA a 0". GPA

0 0.000 0.050 0.100 0.1 50

VOLUME (CC)

FIG. 1. Graph of LVDP uersus left ventricular balloon volume. Data are for control hearts (A), hearts from 8-GBA-fed animals (O), and for hearts from 8-GPA-fed rats (0). The heart rates were 240 beats/min for all hearts.

TABLE I Functional and oxygen consumption data

Functional data were obtained from control and creatine depleted rat hearts under conditions presented in Fig. 1. Measurements were made of LVDP, rates of coronary flow (CF), heart rate (HR) and the rate X pressure product (RXP) an index of contractile function in the isovolumic heart preparation. n = 14 for each condition.

LVDP CF HR RXP consumotion 0 2

rnmHg rnllrnin bpm rnrnHgfmin '"$:"/ Control 240 f 8 17.0 f 0.7 226 f 8 54,400 f 2,000 7.5 f 0.6 GBA 155 ? 9 15.8 f 0.8 219 ? 7 34,000 f 2,000 8.7 f 0.5 GPA 141 f 12 16.4 f 0.9 214 f 10 30,174 f 3,000 8.9 f 0.8

were isolated from four control and four 0-GBA hearts. Meas- urements of ADP/O ratios, in the presence of glutamate and malate, were performed. For the control hearts the ADP/O ratio was 2.8 f 0.1 (mean f S.D.) and for the P-GBA hearts was 2.6 f 0.2. These small differences were not statistically significant. Therefore, the observed functional alteration in the P-GBA hearts does not appear to be due to mitochondrial dysfunction.

An alternative could be that the analogue-fed hearts contained mitochondria which were normal in function, but reduced in quantity, The total tissue activity and the mitochondrial-specific activity of cytochrome oxidase was determined in five control and five P-GBA fed hearts. From these data, one can calculate the mitochondrial content of the tissue, since cytochrome oxidase is a specific mitochondrial marker enzyme. Control hearts contained 74.6 f 11.8 (mean f S.D.) mg of mitochondrial protein/g wet weight of tissue, while hearts from P-GBA treated animals contained 83.4 f 14.4 mg of mitochondrial protein/g of tissue. This slightly higher value in the P-GBA hearts was not significantly different from control and close to values previously reported by Scarpa and Graziotti (32). Therefore, the contractile differences noted in Fig. 1 are not reflected by defects in the mitochondrial parameters we measured.

Electron Microscopy-Thin section electron micrographs were examined to determine if analogue feeding induced any abnormalities in cell structure which might give rise to altered contractile function. The ultrastructure of the myocardium in both control, P-GPA, and B-GBA (data not shown) groups were generally very similar. The morphology of control hearts is shown in Fig. 2 and appears quite normal. As seen in Fig. 3, both the mitochondria and myofibrils appeared morpholog- ically normal in most of the areas of myocardium from P-

GPA-treated animals. On extensive examination (Fig, 4), only rare focal areas of abnormality were seen in the P-GPA tissues with some mild loss of myofibrils and dilated mitochondria. However, in controls similar rare focal areas of abnormality were also seen. Therefore, the myocardial tissue in all groups could not be distinguished on the basis of morphological differences.

Metabolic Characterization NMR Measurements of High Energy Phosphates-In order

to assess the extent of bioenergetic alterations in the creatine- depleted hearts, nuclear magnetic resonance experiments were performed to measure the concentrations of high energy phosphates in the intact heart. With the volume of the left ventricular balloon fixed at the volume yielding maximum LVDP, NMR spectra were obtained to assess the extent of PCr depletion as well as the concentrations of ATP and inorganic phosphate, which appeared normal in control hearts (Fig. 5, upper spectrum). In contrast, in hearts from ,f3-GBA- fed animals (Fig. 5, botton spectrum) the intensity of the PCr resonance was significantly decreased. The data in Table I1 present the quantitative results. A mean depletion of 80% was noted from a concentration of 12.4 pmol/g cell water in control hearts to 2.6 pmol/g cell water in the 14 P-GBA-depleted hearts. The ATP concentration was determined for these two sets of hearts from the integrated area of the P-ATP peak, and concentrations of 9.0 f 0.9 and 9.7 f 0.9 pmol/g cell water were obtained in the control and B-GBA hearts, respectively. The inorganic phosphate concentration was 4.4 f 0.4 in the control group and 4.9 f 0.5 in the 8-GBA group. The intracellular pH determined from the chemical shift of the Pi resonance was 7.17 f 0.02 for the control group and 7.22 & 0.02 in the P-GBA group. It is important to note that in the NMR spectra of the P-GBA hearts, no phosphorylated GBA resonance was observed. This is in marked contrast to spectra observed with similar feeding of P-GPA, in which a large superimposed phosphorylated P-GPA resonance is observed (Fig. 5, middle spectrum).

The absence of a such a large phosphorylated analogue resonance in the P-GBA hearts greatly simplified the process of quantitating the PCr concentration, as well as the process of performing the saturation transfer measurements described below. For these reasons, saturation transfer experiments were conducted only on hearts from either control or 8-GBA- fed animals.

Saturation Transfer Experiments-After the initial control spectra were obtained, saturation transfer experiments were performed in which three separate files were sequentially accumulated. The first was control saturation with the decoupler frequency displaced to the left of the PCr resonance. The second was acquired with saturation of the y-ATP resonance, while the third was with saturation of the PCr resonance (Figs. 6 and 7). In these experiments the length of the effective t+m saturation pulse was 4.0 s. As demonstrated previously, this value was sufficiently long to result in a maximum loss of PCr or y-ATP magnatization upon saturation of the y- ATP or PCr resonances, respectively (12). In order to nor- malize for any minor temporal variation which might occur in each heart preparation, 50 transients were acquired sequentially in each of the three files, with cyclic repetition until a total of 300 transients were obtained.

From these experiments, the value of Mo/Mt- were obtained, Table 111. Separate studies were performed to measure the TI values in the absence of chemical exchange in a group of four control and four p-GBA hearts. Variable duration of saturation of the y-ATP resonance was performed, ranging

Effects of Phosphocreatine Depletion on Heart Bioenergetics 20299 l C r a l l Q {WT" " ." . , . , *y,1","

FIG. 2. Electron micrograph of myocardium from a control rat shows myocytes with normal morphological features. The gluteraldehyde-fixed tissue was Araldite embedded, stained with uranyl acetate, and counterstained with lead citrate as described under "Materials and Methods." The scale bar is 1 DM.

FIG. 3. Electron micrograph of myocardium from a 8-GPA-treated (8 week) rat heart. It shows no significant morphological alterations from control tissue. The shape of an occa- sional mitochondrion is only slightly dis- torted (arrows). Tissue was process as described in Fig. 2 and under "Materials and Methods." The scale bar is 1 pM.

from 0.2 to 4 s. The TI values for PCr and Pi were calculated from the plot of In (MAt - MAL-), as described previously (12). Values of 3.5 & 0.1 and 1.9 f 0.1 s were obtained, respectively, for PCr and Pi in both groups. The TI of the y- ATP was similarly measured with saturation of the PCr resonance and a value of 1.2 & 0.1 s was obtained for either the control or GBA hearts. Utilizing the values of Mo/Mt- and TI determined from the saturation of the y-ATP, the rate constant was calculated for the creatine kinase reaction and for ATP synthesis. The forward rate constant, K, of the creatine kinase reaction was 0.88 s" in the control and 1.17 s" in the p-GBA hearts, Table 111. The flux from PCr to y- ATP calculated from the product of the [PCr] and K was 10.9 pmol/s/g cell water in the controls and 3.0 pmol/s/g cell water in the p-GBA hearts. The reverse rate constant of the creatine kinase reaction, K-', was calculated to be 0.60 s-' in the

controls and 0.15 s-l in the p-GBA hearts. As a result, in both cases the reverse fluxes are lower than forward flux rates, an observation previously seen in our laboratory and by others, which can be accounted for in part by contributions from the myokinase reaction (33). The rate constant for ATP synthesis from ADP was 0.25 s" in the controls and 0.22 s-l in the p- GBA hearts, yielding almost equivalent flux rates.

Measurements of Creatine Kinase-It is possible that these flux differences could reflect a slow adaptational change in the creatine kinase content of the hearts, which occurred during the 8 weeks of analogue feeding. Therefore, measurements of total tissue homogenate and mitochondrial creatine kinase activity were performed in four control and four 0- GBA hearts. The whole heart homogenate creatine kinase activity was 870 2 20 and 710 & 50 IU/g wet weight of tissue in control and p-GBA homogenates, respectively. These re-


FIG. 4. Cross-section of myocardium following 8 weeks of P-GPA treatment. A myocyte shows disarray of myofilaments and focal thickening of Z-lines. Tissue was processed as described in Fig. 2 and under "Materials and Methods." Scale bar is 1 PM.

TABLE I1 Metabolite concentrations

Intracellular metabolite concentrations derived from NMR spectra presented in Fig. 5. ATP free energy calculations were made as described under "Discussion" and previously published (12).

1 " " l " " I " " l " 10 0 - 10 -20 PPM

FIG. 5. 31P NMR spectra of control, /3-GPA, and /3-GBA hearts, top to bottom, respectively. The major resonance peaks are from left to right: Pi, 4.99 ppm; PCr, 0.0 ppm; ?-ATP, -2.5 ppm; n-ATP, -7.6 ppm; @-ATP, -16.1 ppm; and Mg2"trimetaphosphate, -20.1 ppm. These spectra were obtained with 6O"-pulse and 5-s interpulse delay.

sults indicate that the total homogenate creatine kinase activity was decreased in the P-GBA group. The creatine kinase activity in the mitochondrial fraction was 5.65 f 0.5 and 4.35 k 0.5 IU/mg protein in the control and 8-GBA hearts, respectively. Assays of MDH were performed and activities of 13.9 f 1.4 and 8.8 f 0.6 IU/mg protein were obtained in the

ADP Pi ATP Cr PCr IH'I AG prnolfg cell water kcallrnol

Control 0.118 4.4 9.0 18.3 12.4 6.76 X 10"' -13.7 GBA 0.500 4.9 9.7 13.2 2.56 6.03 X -12.8

control and @-GBA hearts. The normalized CK/MDH ratios were 0.406 in the control mitochondria and 0.494 in the P- GBA mitochondria. In spite of this apparent decrease, when one considers the differences in mitochondrial content, total CK homogenate activity, and the mitochondrial CK-specific activity, the content of total CK associated with the mitochondrial fraction is remarkably similar in the two groups, 48.4% in control versus 51.1% in P-GBA hearts.

Measurements of Cellular Creatine and P-GBA-In 12 control and 12 P-GBA hearts measurements of total Cr (Cr + PCr) and P-GBA concentrations were performed upon completion of the NMR experiments. The total Cr concentration was 30.7 f 1.8 (mean * S.D.) pmol/g cell water in the control hearts and 15.8 f 1.4 pmol/g cell water in the GBA hearts. The P-GBA concentration was 12.8 pmol/g cell water in the 0-GBA hearts.

In Vitro Mitochondrial Creatine Kinase Kinetics-Mito- chondria were isolated from four different control hearts, and oxygen consumption measurements were performed with different Cr and P-GBA concentrations in order to determine the apparent K, of the mitochondrial creatine kinase for Cr as well as the K,,, and KI for P-GBA. Duplicate measurements were performed on each of these four mitochondrial preparations. In line with previous reports, the K, for Cr was 5.4 mM. No stimulation of oxygen consumption was observed with P-GBA, even at concentrations of up to 200.0 mM, suggesting that it is not readily phosphorylated by creatine kinase. P-GBA, however, did exhibit weak competitive inhi- bition a t high concentrations with a calculated KI of 94 mM.

Creatine Repletion Experiments-In order to confirm that


A

B

C

C

I I 1 I I I I I

I O 5 0 -5 - 1 0 -15 -20 -25 I I

PPM

FIG. 6. Saturation transfer NMR spectra of control heart. A , saturation to the left of the PCr resonance, a t a frequency offset equal to that from the ?-ATP to the PCr peak. This is a control spectrum. B, saturation of the y-ATP resonance. C, saturation of the PCr resonance. The chemical shift assignments are the same as noted in Fig. 5.

the functional depression observed in the @-GBA hearts was related to phosphagen depletion, experiments were performed to replete cellular phosphocreatine. After completion of the saturation transfer experiments, five control hearts and five P-GBA hearts were perfused with medium containing 50 mM creatine. Serial 10-min NMR acquisitions were obtained, and LVDP and heart rate were recorded. Perfusion of the control hearts with creatine resulted in no significant changes in function (Fig. 8) or in the high energy phosphate content of the hearts. In marked contrast, in P-GBA hearts a 28 f 5% increase in LVDP and a 32 f 6% increase in the rate pressure product was observed during the first 20 min of creatine perfusion (Fig. 9). Likewise, as seen in Fig. 10, by 20 min there also was a significant increase in PCr concentration. This change was approximately a 52% increase from 2.4 to 3.6 Mmol/g cell water. The concentration of ATP, however, remained unchanged. Thus, creatine perfusion in GBA hearts lead to both functional and metabolic recovery.

DISCUSSION

In this study we have observed that depletion of Cr and PCr by feeding Cr analogues markedly altered the contractile

PPM

FIG. 7. Saturation transfer NMR spectra of j3-GBA-treated heart. A, saturation to the left of the PCr resonance, at a frequency offset equal to that between the y-ATP and PCr. B, saturation of the ?-ATP resonance. C, saturation of the PCr resonance. The chemical shift assignments are the same as noted in Fig. 5.

TABLE 111 Saturation transfer data

n = 10 for each condition. control or B-GBA. cw. cell water. ~~ ~

M,,/M,,, TI K [PCr] [PJ [ATP] Flux

slg cw s s-1 prnollg cw prnolJ

ATP saturation PCr .--) ATP

Control 4.09 3.5 0.88 12.4 10.9 GBA 5.10 3.5 1.17 2.6 3.0

Control 1.37 1.5 0.25 4.4 1.10 GBA 1.35 1.6 0.22 4.9 1.08

Control GBA

1.71 1.2 0.60 9.0 5.4 1.18 1.2 0.15 9.7 1.5

ADP + Pi -+ ATP

PCr saturation (ATP .--) PCr)

function and bioenergetic status of the perfused rat heart. Left ventricular pressure volume curves demonstrated that at any given left ventricular volume the left ventricular-developed pressure was much lower in both @-GBA and P-GPA hearts than in controls. In addition, the maximum observed left ventricular-developed pressure was 35% lower than in comparable controls, and the rate pressure product was 37% lower. These differences were highly statistically significant,


50 mM C r J.

0 TIME (minl

35

FIG. 8. Left ventricular pressure and rate recording for a control heart perfused with perfusate containing 50 mM Cr. At time 0 the perfusate was changed from the normal Cr-free perfusate to one containing 50 mM Cr. The units of dP/dt are mmHg/min (uncalibrated).

dP d t -

7 ?-5J-- xL~iXL!!~:L.:I . 1 i i l ,~. A, L- -, L ~ 1 - . , . . . . . . . . . . . .

0 30 TIME (min l

FIG. 9. Left ventricular pressure and rate recording for a &GBA heart perfused with perfusate containing 50 mM Cr and 0.25 mM inorganic phosphate. To minimize the possibility of intracellular phosphate depletion upon creatine phosphorylation, Pi was also added to this perfusate. At time 0 the perfusate was changed to one containing these additional metabolites.

A I

B I

10 0 -10 -20

PPM

FIG. 10. 31P NMR spectrum of a 8-GBA heart before and 20 min after perfusion with 50 mM creatine and 0.25 mM inorganic phosphate. The chemical shift assignments are the same as in Fig. 5.

p < 0.001, and in agreement with previously reported cardiac functional defects in P-GPA-fed rats (34, 35). In spite of the marked reduction in contractile function in the 0-GBA or @-

GPA hearts, the oxygen consumption was actually slightly increased. Electron microscopy studies of the morphology and ultrastructures of these hearts demonstrated normal mitochondrial and myofibilar structure. The myocardium of these hearts could not be distinguished from the controls even at the ultrastructural level. 31P NMR studies demonstrated that 8-GBA feeding resulted in an 80% PCr depletion. As shown in Table 111, in control animals the PCr concentration was 12.4 and in the GBA group it was 2.5 pmol/g cell water. In the 8-GBA animals, small but statistically significant differences were noted in the Pi and ATP concentrations with 11% increase in Pi and 8% increase in ATP. In addition, the pH calculated from the chemical shift in the Pi resonance was significantly more basic in the @-GBA animals, 7.22 uersus 7.17. The Cr concentration was also measured from tissue extracts and, as shown in Table 11, the Cr concentration was reduced 28% compared to controls. Therefore, while both PCr and Cr are depleted in these hearts, PCr was considerably more effected.

Based on the more marked PCr depletion as well as the more alkaline pH of the @-GBA hearts the free ADP concentration estimated from the creatine kinase reaction appeared to be considerably elevated. If we assume that the creatine kinase reaction is in the near equilibrium, it is possible to calculate the cellular ADP concentration from the equation:

[ADP] = [ATP] [Cr]/K,[PCr][H+]

where Keg is the equilibrium constant of the creatine kinase reaction. The free M e in heart muscle has recently been estimated to range between 0.48 to 1.6 mM (36). It is known that the Keg has a value of 1.66 X lo9 "' for a M e of 1.0 mM (37). From measurement of the chemical shift values of the @-ATP and comparison to the in vitro observed chemical shift value, it can be inferred that the cellular Mg2+ in both P-GBA and control hearts is in the range 1.0-1.5 mM (38). Inserting the values for ATP, Cr, PCr, H' shown in Table 11, we calculate that the cellular free ADP concentration is 118 PM in control hearts and 500 p~ in the @-GBA hearts. Thus, in the P-GBA hearts there appears to be at least a 5-fold increase in the free ADP concentration. The observed high ADP concentration is even more striking when compared to the typical values observed in glucose-perfused hearts not stressed to perform at near maximum; ADP concentrations of 58 p~ have been reported (12). In both skeletal and cardiac muscle, inverse correlation of ADP concentrations and contractile function have previously been observed (12,39).

In principle, it is possible to directly measure the free ADP concentration from differences in the integrated areas of the 0- and y-ATP peaks since the @-ADP resonance is superimposed on the y-ATP peak while the @-ATP resonance has no other superimposed peaks. Signal to noise ratios for these peaks of up to 25:l were obtained. For the control hearts spectra, no significant difference in the integrated area of these peaks was noted, indicating that the ADP concentration was less than 360 p ~ . In the /3-GBA hearts, however, the area of the y-ATP peak was 12 f 4% greater than the @ATP peak. This would suggest that the actual free ADP concentration might be as high as 780 pM to 1.55 mM. In view of the potential sources of error in peak integration, some caution should be exercised in accepting these calculated ADP concentrations. However, in view of the absence of differences in the P- and y-ATP of identical control spectra as well as in previously analyzed spectra, the observed differences in the 8-GBA spectra appear to be real. These measured ADP concentrations values would suggest that in the @-GBA hearts, where PCr is markedly depleted, the creatine kinase reation


may not be in near equilibrium. This observation is not surprising considering the large concentrations of ADP which must be generated at the myofibrils with each contraction and the 80% decrease in the concentration of PCr in the P- GBA hearts.

A steady-state decline in the concentration of ADP could have a direct influence on the cytosolic phosphorylation potential, which in turn might explain some of the observed differences in contractile function. Therefore, knowing the value of ADP, ATP, and Pi, the in uiuo free energy of ATP hydrolysis was calculated from the equation:

AG = AG” + RT In [ADP] X [Pi]/[ATP]

In the literature different values have been calculated for the AGO at physiologic conditions. Assuming a temperature of 37 “C and [ M F ] of 1.0 mM Veech et al. (37) calculated a value of -7.73 kcal/mol while Lehninger (40) previously calculated a value of -7.30, and Kammermeier et al. (41) more recently a value of -7.24 kcal/mol. Using the AGO value of Veech et al., we calculate that the AG in the control hearts is -13.7 kcal/mol and -12.8 kcal/mol in the P-GBA hearts. Thus, the AG value in the p-GBA hearts is significantly lower than in the control hearts. These values are much lower than the value of -14.7 kcal/mol which we have previously calculated for glucose-perfused hearts at lower work levels (12).

At first glance, the 0.9 kcal/mol change in the free energy of ATP hydrolysis between the control hearts and the GBA hearts may appear to be small. However, this difference could have major consequences if the free energy of ATP hydrolysis approaches the value required for the actin-myosin-ATPase or for ion pump ATPases. Recently, Kammermier et al. (41, 42) has calculated that the sacroplasmic reticulum Ca2+-ATP- ase requires the highest free energy, a value of -12.4 kcal/ mol. This value was calculated using a AGO value of -7.24. If this “limit” is recalculated using the Veech A G value, the limit is -12.87 kcal/mol. Thus, the calculated AG seen in the @-GBA hearts is at, or even below, that required for the sacroplasmic reticulum Ca2+-ATPase. Furthermore, the case becomes even more problematic if we calculate the free energy of ATP hydrolysis using the higher concentrations of ADP derived from the NMR spectra of the P-GBA hearts. Under these conditions, the calculated values are in the range of -12.0 to -11.6 kcal/mol, or even more clearly below the limit. Therefore, one would expect that in the P-GBA hearts the Ca2+-ATPase could be unable to resequester Ca2+ in the sacroplasmic reticulum to maintain the same cellular Ca2+ gradients. In support of this notion, we observed that at the balloon volume yielding maximum left ventricular-developed pressure the diastolic pressure was elevated by 7 mmHg in P- GBA hearts compared to the identical controls. This difference was statistically significant with a p < 0.01. Previously, we have observed that in paired pulse stimulation studies of papillary muscle from control and P-GBA hearts that the fusion of the first and second twitches occurred after a much longer interpulse interval in the p-GBA muscle suggesting delayed Ca2+ resequestration into the sarcoplasmic reticulum (43). The decrease in free energy of ATP hydrolysis thus could account for these previously unexplained observations.

The actin-myosin-ATPase has been estimated to require in the range of 10.7-12 kcal/mol (41). Thus, the decreased free energy of ATP hydrolysis observed in the P-GBA hearts could also be adversely effecting contractile function at the level of the myofibrils themselves. Perhaps the creatine-depleted P- GBA hearts cannot achieve the same high near maximal levels of cardiac function because the elevations in free ADP concentration would be sufficient to drive the AG below the

critical value required to sustain contraction. The low AG values which we calculate at the reduced levels of cardiac function are already on the threshold of this required free energy. This suggests that Cr and PCr depletion decreases the energetic efficiency of the heart. This reduced energetic efficiency could be due to the marked decrease in phosphorylation potential at sites of ATP utilization.

Marked alterations in the in vivo kinetics of the creatine kinase reaction were also observed. The flux of the ATP synthesis from PCr was markedly decreased from 10.9 to 3.0 pmol/s/g cell water in the control and P-GBA hearts, respectively. The flux of ATP synthesis from ADP, however, was approximately the same in both groups, in spite of the 16% increase in oxygen consumption which was observed in the P- GBA hearts. The calculated ATP/O ratios are thus 1.9 k 0.2 and 1.6 f 0.2 in the control and P-GBA hearts, respectively. These values are somewhat lower than the values in the range of 2 to 3 which we have previously observed in perfused hearts at lower work levels. It is possible that in both the control and (3-GBA hearts stressed to function at near maximal work levels that there is some uncoupling of oxidative phosphorylation, perhaps mediated by mitochondrial calcium cycling (44).

In conclusion, these experiments demonstrate that Cr, PCr, and creatine kinase may serve an important function in optimizing the thermodynamic efficiency of cardiac energy metabolism. At the myofibrils where large concentrations of ADP are generated, creatine kinase catalyzes the reaction

PCr + ADP -t ATP + Cr

which serves to lower the ADP concentration, thus increasing the free energy of ATP hydrolysis. At the mitochondria where ATP concentrations are high and ADP concentrations rela- tively low, creatine kinase catalyzes the reaction

ATP + Cr ”-* ADP + PCr

which serves to increase the local ADP concentration avail- able to stimulate mitochondria respiration, as well as to decrease the phosphorylation potential required for ATP synthesis. Thus, compartmentalized creatine kinase serves the dual functions of maximizing the free energy of ATP hydrolysis at sites of utilization such as the myofibrils and sarcoplasmic reticulum, while also minimizing the free energy required for ATP synthesis at the mitochondrion. In this manner, it appears that creatine kinase enables the heart to achieve high levels of sustained contractile function that are not otherwise observed in either depleted (34, 35) or creatine kinase-inhibited hearts (15-17).

Acknowledgments-We wish to acknowledge the expert technical assistance of Koenraad M. Vandegaer, Kathy May, and Lee Shang.

REFERENCES

1. Zweier, J. L., Korecky, B., and Jacobus, W . E. (1986) J. Mol. Cell.

2. Zweier, J. L., Korecky, B., and Jacobus, W . E. (1986) Circulation

3. Brautbar, N. (1986) Adu. Exp. Med. Biol. 194, 1-212 4. Hill, A. V. (1932) Physiol. Reu. 12,56-67 5. Englehardt, W . A,, and Ljubimowa, M. N. (1939) Nature 144,

6. Lipmann, F. (1941) Adu. Enzymol. 1, 99-162 7. Gudbjarnason, S., Mathes, P., and Ravens, K. G. (1970) J. Mol.

8. Saks, V. A., Rosenshtraukh, L. V., Smirnov, V. N., and Chazov,

Cardiol. 18, Suppl. 3, 79

74,II-326

668-669

Cell. Cardiol. 1, 325-339

E. I. (1978) Can. J . Physiol. Pharmacol. 56,691-706

20304 Effects of Phosphocreatine Depletion on Heart Bioenergetics 9. Bessman, S. P., and Geiger, P. J. (1982) Science 2 1 1 , 448-452

10. Bessman, S. P., and Carpenter, C. L. (1985) Annu. Reu. Biochem.

11. Jacobus, W. E. (1985) Annu. Reu. Physiol. 47,707-725 12. Zweier, J. L, and Jacobus, W. E. (1987) J. Biol. Chem. 2 6 2 ,

13. Jacobus, W. E. (1985) Biochem. Biophys. Res. Commun. 133,

14. Jacobus, W. E., and Diffley, D. M. (1986) J. Biol. Chem. 2 6 1 ,

15. Fossel, E. T., and Hoefeler, H. (1985) SOC. Mag. Reson. Med. 4,

16. Hamman, B. L., Bittl, J. A., Jacobus, W. E., and Ingwall, J. S.

17. Fossel, E. T., and Hoefeler, H. (1986) Circulation 7 4 , 11-326 18. Meyer, R. A., Sweeney, H. L., and Kushmerick, M. J. (1984) Am.

J. Physiol. 246 , C365-C377 19. Meyer, R. A., Brown, T. R., and Kushmerick, M. J. (1984)

Biophys. J. 45,91a 20. Shoubridge, E. A., and Radda, G. K. (1984) Biochim. Biophys.

Acta 806,79-88 21. Shoubridge, E. A., Jeffry, F. M. H., Keogh, J. M., Radda, G. K.,

and Seymour, A. M. L. (1985) Biochim. Biophys. Acta 847,25- 32

22. Rowley, G. L., Greenleaf, A. L., and Kenyon G. L. (1971) J. Am. Chem. SOC. 93,5542-5551

23. Saks, V. A., Kupriyanov, V. V., Elizarova G. V., and Jacobus, W.

24. Jacobs, E. E., Jacob, M., Sanadi, D. R., and Bradley, L. B. (1956)

25. Saks, V. A., Charnousova, G. B., Gukovsky, D. E., Smirnov, V.

26. Schnaitman, C., and Greenawalt, J. W. (1968) J. Cell. Biol. 38,

27. Jacobus, W. E., Pores, I. H., Lucas, S. K., Kallman, C. H., Weisfeldt, M. L., and Flaherty, J. T. (1982) in ZntracellularpH:

54,831-862

8015-8021

1035-1041

16579-16583

655-656 (abstr.)

(1986) SOC. Mag. Reson. Med. 5, WP 133-134 (abstr.)

E. (1980) J. Biol. Chern. 255, 755-763

J. Biol. Chem. 223 , 147-156

N., and Chazov, E. I. (1975) Eur. J. Biochem. 57,273-290

158-175

Its Measurement, Regulation and Utilization in Cellular Func- tions (Nuccitelli, R., and Deamer, D. W., eds) pp. 537-565, Alan R. Liss, Inc., New York

28. Dubnoff, J. W. (1957) Methods Enzymol. 3,635-650 29. Bonas, J. E., Cohen, B. D., Natelson, S. (1963) Microchem. J. 7,

30. Eppenberger, H. M., Dawson, D. M., and Kaplan, N. 0. (1967)

31. Ochoa, S. (1955) Methods Enzyrnol. 1 , 735-739 32. Scarpa, A., and Grazotti, P. (1973) J. Gen. Physiol. 62, 756-772 33. Ugurbil, K. (1985) J. Mag. Reson. 64,207-219 34. Kapelko, V. I., Kupriyanov, V. V., Novikova, N. A., Lakomkin,

V. L., Steinschneider, A. Ya., Severina, M. Yu., Veksler, V. I., and Saks, V. A. (1988) J. Mol. Cell. Cardiol. 20,465-479

35. Mekhfi, H., Hoerter, J., Lauer, C., Wisnewski, C., Schwartz, K., and Ventura-Clapier, R. (1990) Am. J. Physiol. 2 5 8 , H1151- H1158

36. Murphy, E., Freudenrich, C. C., Levy, L. A., and London, R. E. (1989) Proc. Natl. Acad. Sci. U. S. A. 8 6 , 2981-2984

37. Veech, R. L., Randolph-Lawson, J. W., Cornell, N. W., and Krebs, H. A. (1979) J. Biol. Chem. 254,6538-6547

38. Kushmerick, M. J., Dillon, P. F., Meyer, R. A., Brown, T. R., Krisanda, J. M., and Sweeney, H. L. (1986) J. Biol. Chem. 2 6 1 ,

39. Dawson, M. J., Gadian, D. G., and Wilkie, D. R. (1978) Nature

40. Lehninger, A. L. (1975) Biochemistry, pp. 398-400, Worth Pub-

41. Kammermeier, H., Schmidt, P., and Jungling E. (1982) J. Mol.

42. Kammermeier, H. (1987) Basic Res. Cardiol. 82, Suppl. 2, 31-36 43. Korecky, B., and Brandejs-Barry, Y. (1987) Basic Res. Cardiol.

8 2 , (Suppl. 2) 103-110 44. Hoerter, J. A., Miceli, M. V., Renlund, D. G., Jacobus, W. E.,

Gerstenblith, G., and Lakatta, E. G. (1986) Circ. Res. 58,539- 551

63-77

J. Biol. Chem. 242 , 204-209

14420-14429

274,861-866

lishers, Inc., New York

Cell. Cardiol. 14, 267-277

bioenergetic consequences of cardiac phosphocreatine depletion

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