Failure to Maintain a Low ADP Concentration Impairs Diastolic Function in Hypertrophied Rat Hearts
Background Mechanisms in addition to diastolic calcium overload may contribute to diastolic dysfunction in hypertrophied hearts. In this study, we tested the hypothesis that failure to maintain a low ADP concentration in hypertrophied hearts contributes to diastolic dysfunction by inhibiting the rate of cross-bridge cycling.
Methods and Results By perfusing isolated rat hearts with pyruvate and 2-deoxyglucose (2DG), we were able to perturb [ADP] with minimal changes in [ATP] and [inorganic phosphate] or the contribution of glycolytic ATP to ATP synthesis. The effects of 2DG were compared in aortic-banded (LVH, n=5) and sham-operated (control, n=5) rat hearts. 31P NMR spectroscopy was used to measure the concentrations of phosphorus-containing compounds. We found a threefold increase of left ventricular end-diastolic pressure (LVEDP) in LVH during 2DG perfusion, and this increase was concomitant with a threefold increase in intracellular free [ADP]. The [ADP] in the control hearts was maintained <40 μmol/L, and no change in LVEDP was observed. A linear relationship between increases in [ADP] and LVEDP was found (r2=.66, P=.001). Furthermore, the capacity of the creatine kinase reaction, a major mechanism for maintaining a low [ADP], was decreased in LVH (P=.0001).
Conclusions Increased [ADP] contributes to diastolic dysfunction in LVH, possibly due to slowed cross-bridge cycling. Decreased capacity of the creatine kinase reaction to rephosphorylate ADP is a likely contributing mechanism to the failure to maintain a low [ADP] in LVH.
Hypertrophied hearts exhibit prolonged diastolic relaxation and greater diastolic dysfunction than normal adult hearts during energy deprivation.1 2 3 It has been suggested that the susceptibility to impaired relaxation is due to diastolic calcium overload in these hearts.1 2 3 4 However, a recent study showed a greater impairment of relaxation during energy deprivation in myocytes isolated from hypertrophied hearts than in controls, even though the diastolic calcium level increased to a similar extent for both groups.5 These results show that it is very likely that mechanisms in addition to diastolic calcium overload also contribute to the impairment of relaxation in hypertrophied myocardium.
The rate of actomyosin cross-bridge cycling, directly responsible for force generation and relaxation, is controlled by the concentrations of substrates and products of the actomyosin ATPase reaction: ATP, ADP, and Pi. The release of ADP has been shown to be a rate-limiting step for cross-bridge dissociation in cardiac and skeletal muscle fibers.6 7 Thus, an increase in free [ADP] should inhibit the rate of ADP release from the actomyosin complex and thereby reduce the rate of cross-bridge cycling. If this were to occur, myofibrillar relaxation would be impaired. Under physiological conditions, several mechanisms ensure that the free [ADP] is maintained at a low level in myocytes. Among them, the CK reaction plays a prime role. This is because the rate of ATP turnover via this reaction is at least an order of magnitude higher than that of oxidative phosphorylation, glycolysis, or the adenylate kinase reaction.8 By rapidly transferring a phosphoryl group from PCr to ADP via the CK reaction (PCr+ADP+H+ ⇔ ATP+Cr), the free [ADP] is normally maintained in a low range despite the fluctuations in the rate of ATP synthesis and utilization. Previous studies showed that the activity of CK and the total content of Cr in severely hypertrophied and failing hearts are significantly reduced, indicating a decreased capacity of this reaction.9 10 11 This decrease may compromise the ability of hypertrophied myocardium to maintain a low intracellular free [ADP], especially under stress conditions.
This study was designed to test the hypothesis that failure to maintain a low [ADP] may render the hypertrophied heart more susceptible to diastolic dysfunction. Fig 1⇓ illustrates our strategy to perturb [ADP] with minimal changes in [ATP] and a low [Pi] in an isolated perfused rat heart preparation. We first perfused the hearts with a buffer containing pyruvate and glucose (Fig 1⇓, left) and then switched to another buffer in which glucose was substituted with 2DG. Because 2DG is not an appropriate substrate for the glycolysis, it accumulates as 2DG-P after entering the cell (Fig 1⇓, right). Phosphorylation of 2DG requires energy from ATP hydrolysis, and accumulation of 2DG-P traps free intracellular Pi. As a result, the intracellular [ATP] and [Pi] should decrease and free [ADP] should increase. Since [ADP] is in the micromolar range and [ATP] is at the millimolar level, a significant increase of [ADP] can occur with only a slight decrease of [ATP]. By supply of a large amount of pyruvate in the perfusate (5 mmol/L), ATP synthesis via oxidative phosphorylation in these hearts was sustained. Under these conditions, ATP synthesis via glycolysis was trivial (<1% of total ATP production).12 Therefore, perturbing glycolysis by supplying 2DG to these hearts did not substantially decrease ATP synthesis, and normal [ATP] was maintained. Using this strategy, we found that an increase in free [ADP] is closely related to an increase in LVEDP in isolated rat hearts, and the greater increase of [ADP] in hypertrophied hearts than in the control hearts may be one mechanism responsible for the enhanced susceptibility to developing diastolic dysfunction in these hearts.
Animal Model of LVH
Weanling male Wistar rats (body weight, 75 to 100 g) were purchased from Charles River Breeding Laboratories (Wilmington, Del). LVH was induced by banding the ascending aorta as previously described.5 Briefly, the animal was anesthetized with intraperitoneal pentobarbital, the chest was opened, a stainless steel clip was placed on the ascending aorta, and the chest wall was quickly closed. The sham-operated animal was subjected to the same operation procedure but without the clip. Animals were fed normal rat chow and water ad libitum and were used 39 weeks after banding.
Isolated Perfused Heart Preparation and Experimental Protocol
Rats were anesthetized by 50 mg sodium pentobarbital IP. The chest was opened, and the heart was rapidly excised and arrested in ice-cold buffer. The heart was immediately attached to a perfusion apparatus, and retrograde perfusion via the aorta was carried out at a constant coronary flow at 37°C. The flow was adjusted to achieve mean perfusion pressures of 80 and 110 mm Hg for control and LVH hearts, respectively. These perfusion pressures were chosen in recognition of the difference between the in vivo coronary perfusion pressures of the LVH and the control hearts. Prior experience showed that this approach would achieve comparable myocardial flow rates per gram of LV weight for the two groups.3 13 Immediately after the perfusion began, the root of the pulmonary artery was cut open to allow for the right ventricular outflow. The flow of the thebesian veins was drained by a thin polyethylene tube pierced through the LV apex. A water-filled latex balloon was inserted into the left ventricle through an incision in the left atrium and was connected to a pressure transducer (Viggo-Spectramed P23XL) for continuous recording of LV pressure. LVEDP was set to 10 mm Hg by adjustment of the volume of the balloon. Thereafter, balloon volume was held constant throughout the protocol. All hearts were paced at 4.5 Hz by a Grass stimulator (Grass Instrument Co). The heart was surrounded by its own perfusate in a glass NMR tube, and the perfusate level was kept just above the left atrium by continuous suction through a polyethylene tube.
All hearts were first perfused with insulin-free Krebs-Henseleit buffer of the following composition (mmol/L): NaCl 118.0, KCl 3.5, CaCl2 1.75, MgSO4 1.2, KH2PO4 1.2, EDTA 0.5, NaHCO3 25, pyruvate 5.0, and glucose 5.0 (equilibrated with 95% O2/5% CO2, pH 7.4) and stabilized for 15 to 20 minutes. After a baseline measurement, hearts were switched to a 2DG (Sigma Chemical Co) buffer in which glucose was substituted with 5 mmol/L of 2DG. All hearts were perfused with 2DG buffer for an additional 20 minutes. At the end of each experiment, the heart was weighed, frozen, and stored at −80°C for biochemical assays.
31P NMR Spectroscopy
Intracellular concentrations of PCr, ATP, Pi, and 2DG-P were measured by 31P NMR spectroscopy. Spectra were obtained at 161.94 MHz on a GE-400 Omega spectrometer. The heart was placed in a 20-mm NMR sample tube inserted into a broad-band probe situated in an 89-mm-bore, 9.4-T superconducting magnet. Spectra were collected without proton decoupling at a pulse width of 27 μs, 60° pulse angle, recycle time of 2.3 seconds, and sweep width of 6000 Hz. Spectra were analyzed with 20-Hz exponential multiplication and zero- and first-order phase corrections. Resonance peaks were fitted to a lorentzian function, and the areas under the peaks were calculated by the commercially available program NMR1 (NMRi). By comparison of the peak areas of fully relaxed (recycle time, 10 seconds) with those of partially saturated (recycle time, 2.3 seconds) spectra, the correction factors for saturation were calculated for [β-P]ATP (1.0), PCr (1.2), Pi (1.15), and 2DG-P (1.35). The baseline NMR spectrum was collected after the stabilization period by averaging signals from 520 free induction decays. During the 20-minute perfusion with 2DG, NMR measurements were made every 2 minutes by averaging of signals from 52 free induction decays.
Ventricular tissue (5 to 10 mg) was thawed and homogenized for 10 seconds at 4°C in potassium phosphate buffer containing 1 mmol/L EDTA and 1 mmol/L β-mercaptoethanol, pH 7.4 (final concentration of 5 mg tissue/mL). Aliquots were removed for assays of protein by the method of Lowry et al14 with BSA as the standard and assays of total Cr content by a fluorometric assay.15 Triton X-100 was then added to the homogenate at a final concentration of 0.1% for analysis of the total CK activity.16 The CK activities were measured at 30°C and are expressed as international units (IU=μmol/min) per milligram of cardiac protein. All reagents used were at least analytic grade and were obtained from Sigma Chemical Co.
The ATP content of the isolated perfused rat heart at the end of the stabilization period has been determined to be 31.1±1.8 nmol/mg protein.17 By use of a value of 0.155 mg protein/mg blotted wet tissue and the reported value of 0.48 μL intracellular water/mg blotted wet tissue,18 [ATP] was calculated to be 10.0 mmol/L. Therefore, the [β-P]ATP peak area of the NMR spectrum obtained at baseline was normalized by the heart weight and was set to 10.0 mmol/L for the control group. The [PCr], [Pi], and [2DG-P] for both control and LVH hearts and [ATP] for LVH hearts were calculated from the ratios of their peak areas per gram heart weight to that of [β-P]ATP area per gram heart weight for the controls. pHi was determined by comparison of the chemical shift of Pi and PCr in each spectrum with values from a standard curve.
Cytosolic free [ADP] was calculated from the equilibrium constant of the CK reaction19 and values obtained by NMR spectroscopy and biochemical assay: [ADP]=([ATP][free Cr])/([PCr][H+]Keq), where Keq=1.66×109 (mol/L)−1 at pH 7.0 and free [Mg2+]=1.0 mmol/L.
The product of myocardial total Cr concentration and total CK activity (Vmax) was used to estimate the maximal CK reaction velocity in vivo, which represented the capacity of energy reserve via this reaction20 : Capacity of the CK reaction=Vmax[total Cr].
All results were expressed as mean±SEM. Measurements made before and during 2DG perfusion were compared by repeated-measures ANOVA for each group. Comparisons between the two groups at the time points of interest were performed by unpaired t test. All the statistical analyses were performed with Statview (Brainpower Inc), and a value of P<.05 was considered significant.
Five controls and five LVH rats were studied. The body weight was comparable in the LVH and the control groups (670±34 versus 693±33 g, P=.64), whereas the heart weight was markedly increased in the LVH group (2.96±0.22 versus 2.00±0.04 g, P=.002). The heart-weight-to-body-weight ratio was 52% higher in the LVH group (4.44±0.34 versus 2.92±0.14 mg/g, P=.003).
Table 1⇓ shows the baseline LV function of both groups. When the LVEDP was set to 10 mm Hg, the LV volume was not different between the two groups. The LVDP was 54% higher in the LVH group. To estimate the load per unit of myocardium, we assumed a spherical LV for both groups. Since the LV volume was similar (Table 1⇓), the LV radius was comparable for the two groups. By Laplace’s law, the LV wall stress was inversely proportional to the wall thickness, which was directly related to LV mass. Therefore, LVDP was normalized by ventricular weight (LVDP/g) and was used as an approximation of wall stress under our experimental conditions.21 LVDP/g was not different between LVH and control hearts.
High-Energy Phosphate Content and the CK System
Table 2⇓ shows the baseline concentrations of ATP, PCr, Pi, and ADP measured by 31P NMR spectroscopy and the activity of CK and the total Cr content measured in tissue homogenates for LVH and control hearts. In LVH hearts, [PCr] was lower while [Pi] and [ADP] were higher than in controls. [ATP] and pHi were not significantly different in the two groups (Table 2⇓) and did not change throughout the protocol. Total content of Cr but not the activity of CK (Vmax) was decreased in LVH hearts. The capacity of the CK system, estimated by the product of total Cr content and the Vmax of the enzyme, was substantially reduced (by 43%).
Effects of 2-DG on High-Energy Phosphate Metabolism
The accumulation of 2DG-P in hearts of both groups is shown in Fig 2⇓. A significantly higher accumulation was observed in the LVH group (P=.001). Thus, comparisons of the LV function between the two groups were made both at the end of the protocol (after 20 minutes of 2DG perfusion) and at the time points when a similar accumulation of 2DG-P was achieved (5.5±0.9 and 6.7±0.8 mmol/L, P=.306) for both control and LVH groups (indicated by the arrows).
Fig 3⇓ shows the changes in concentrations of ATP, Pi, and ADP during the 20-minute perfusion with 2DG. There was a small but significant decrease in [ATP] in both control (−19%, P=.001) and LVH (−22%, P=.001) hearts, and no difference between the two groups was found during the 20-minute period. The [Pi] was kept low by 2DG perfusion and was not significantly different in the two groups (0.85±0.15 versus 1.40±0.54 mmol/L, P=.364). Free [ADP] increased in both groups by the end of the 20-minute 2DG perfusion. It increased moderately in the controls (from 11 to 20 μmol/L) but substantially in the LVH group and was >100 μmol/L by 20 minutes of 2DG perfusion. At the time points when accumulations of 2DG-P in LVH and control hearts were similar, [ADP] was significantly higher in the LVH group than in the controls (indicated by the arrows in Fig 3⇓, P=.017).
Effect of 2DG on LV Function
Fig 4⇓ shows the LVSP/g and LVEDP in control and LVH hearts during 20-minute 2DG perfusion. The LVSP/g was not different between the two groups either at the baseline (P=.650) or during 2DG perfusion (P=.215). Twenty minutes of 2DG perfusion did not cause any change in LVEDP in the controls (P=.886), whereas it caused a threefold increase in LVEDP in LVH hearts (P=.001). Since it has been postulated that glycolytic intermediates might play a role in inhibiting relaxation, hearts were also compared at times of equal accumulation of 2DG-P. An increased LVEDP was observed in LVH hearts compared with the controls (arrows in Fig 4⇓, P=.005). Thus, an increase in LVEDP did not correlate well with the increase in 2DG-P.
Relationship of [ADP] and LVEDP
In Fig 5⇓, LVEDP before and during 20 minutes of 2DG perfusion was plotted against the [ADP] obtained at the same time point for each individual heart. By use of all data points of all hearts, a linear relationship was obtained between the increase of LVEDP and [ADP] (LVEDP=0.23[ADP]+5.76, r2=.66, P=.001). Note that [ADP] was maintained at a level <40 μmol/L in all control hearts, whereas [ADP] in LVH hearts increased up to a concentration of 150 μmol/L. There was no relationship between LVEDP and LVDP/g, [ATP], or [Pi].
The contribution of decreased high-energy phosphate content to diastolic dysfunction has been well documented during ischemia and hypoxia.2 22 These studies, however, were not able to distinguish the effects of ATP depletion, ADP accumulation, or Pi accumulation on myocardial relaxation. Using a moderate dose of 2DG to perturb intracellular energy metabolism in isolated perfused rat hearts, we have successfully increased intracellular free [ADP] while maintaining a low [Pi] and relatively high [ATP]. This occurs because phosphorylation of 2DG requires hydrolysis of ATP to ADP and Pi, and accumulation of 2DG-P traps free intracellular Pi. Because the ATP synthesis via oxidative phosphorylation in these hearts was sustained by the presence of pyruvate and the amount of 2DG entered into the cell is relatively low in the absence of insulin in the perfusate, the [ATP] decreased only slightly. Importantly, [ATP] decreased to a similar extent in both control and hypertrophied hearts during 2DG perfusion. Thus, this strategy gives us a unique opportunity to test the hypothesis that an increase in intracellular free [ADP] alone is sufficient to cause impairment of myocardial relaxation in LVH.
Relationship Between [ADP] and Diastolic Function
Our results show a close relationship between the increase of intracellular free [ADP] and the increase of LVEDP in isolated isovolumic rat hearts (r2=.66, P=.001). Previous studies using skinned skeletal muscle fibers have shown that the dissociation of ADP from the actomyosin complex is a rate-limiting step in cross-bridge cycling.6 The velocity of actin filaments sliding on cardiac myosin was markedly reduced by high [ADP].7 This decrease was found to be more pronounced for cardiac myosin than for skeletal muscle myosin.7 This observation suggests that the intact heart would have a high susceptibility for diastolic dysfunction when [ADP] increases. We have recently shown that an increase in intracellular free [ADP] was closely correlated with the elevated LVEDP in well-oxygenated normal hearts with intact glycolysis and unchanged [Pi].23 Taken together, these results strongly indicate that the contribution of an increased free [ADP] to the impairment of myocardial relaxation, which has been suggested by studies using isolated contractile proteins or skinned muscle fibers, also applies to the intact rhythmically beating heart.
Furthermore, recent studies on smooth muscle fibers indicate that the effect of increased [ADP] on the conformation of myosin differs from that of ATP depletion.24 Thus, it is likely that independent mechanisms are responsible for the impairment of relaxation of muscle fibers caused by increased free [ADP] versus depletion of [ATP]. This supports the previous and current observations that diastolic dysfunction in intact hearts can be caused by marked increase in free [ADP] even at a high [ATP].23 We suggest that diastolic dysfunction caused by an increase of free [ADP] may be mechanistically distinct from the myocardial contracture observed in the rigor state due to decreased [ATP].
[ADP] in Hypertrophied Hearts
Previously, the increased susceptibility of hypertrophied hearts to development of diastolic dysfunction during metabolic stress has been attributed to increased intracellular calcium concentration.1 2 3 The results presented here show that increasing the free [ADP] also impairs diastolic function. Our results suggest that hypertrophied hearts have a reduced ability to maintain intracellular free [ADP] in a low range, which renders these hearts more susceptible than normal hearts to developing diastolic dysfunction. This finding in the intact heart is consistent with prior observations that exposure to 2DG caused markedly slowed relaxation and incomplete relengthening in isolated hypertrophied myocytes from aortic-banded rats compared with control rats despite comparable mild elevation of diastolic calcium levels.5
Role of the CK System
In the well-oxygenated heart, the intracellular free [ADP] is rigorously maintained in the low micromolar range. The precise value is determined in part by the carbon substrate used to synthesize ATP. Free [ADP] is always many orders of magnitude lower than intracellular [ATP] (10 mmol/L). Among other mechanisms, the CK reaction plays a critical role in maintaining this very high ATP-to-ADP ratio by rapidly transferring a phosphoryl group between PCr and ATP. This is due to the high abundance of the enzyme and to the rapid reaction velocity (an order of magnitude higher than oxidative phosphorylation).8 In the present study, we found that the capacity for the CK reaction, estimated as the product of Vmax and total [Cr], was substantially lower in hypertrophied hearts (decreased by 43%). This decreased capacity of the CK reaction may contribute to the impaired ability of hypertrophied hearts to maintain the [ADP] as low as that of the controls. Results presented here show that free [ADP] was higher in LVH hearts than in controls both during baseline conditions and during 2DG perfusion even when accumulation of 2DG-P was similar in the two groups. This observation is consistent with our previous findings that acutely inhibiting the CK activity in hearts with normal Cr content resulted in increases in free [ADP] and LVEDP.23
Using 2DG perfusion as a tool to manipulate ADP levels, we found that the capacity of the CK system for maintaining a low [ADP] was decreased in hypertrophied hearts. This decrease may contribute to the development of diastolic dysfunction in these hearts. Because of the ubiquitous nature of hypertrophy and diastolic dysfunction in the evolution of heart failure, our findings have potential clinical significance. Changes in the CK system can be found during the development of heart failure due to many different kinds of cardiac pathological conditions.11 20 25 Previous studies in hypertrophied and failing human myocardium have shown changes in the CK system25 26 27 and in LV diastolic function28 29 similar to those reported here. Recent studies using animal models of cardiac hypertrophy and failure have shown that changes in the CK system of hypertrophied and failing hearts occurred much earlier than any significant change in [ATP].11 30 31 Furthermore, we recently showed that decreased energy reserve via the CK system could be found in an early stage of cardiac dysfunction when baseline function was normal but contractile reserve was reduced.20 The findings of these prior studies and the present experiments are consistent with the clinical observation that impaired diastolic function may occur early in cardiac hypertrophy, sometimes much earlier than the development of systolic dysfunction.28 29 Thus, a reduced capacity to maintain a low [ADP] may be an early characteristic of hypertrophied and failing myocardium, which contributes to further worsening of cardiac function.
Limitations of the Study
Since the rate of 2DG-P accumulation was faster in hypertrophied hearts than in controls, more 2DG-P accumulated in hypertrophied hearts relative to the controls at the same time point. Therefore, we compared the two groups at two time points: at the end of the 20 minutes of 2DG perfusion and at a time when accumulation of 2DG-P was similar. It would be ideal to match the rate of 2DG-P accumulation in the two groups. However, it is difficult to increase the rate in control hearts without using insulin, which by itself may introduce other confounding factors, such as changes in enzyme activities and metabolic fluxes. In addition, calculating free [ADP] by the CK reaction equilibrium expression provides an average free intracellular [ADP]. It does not provide any information regarding free [ADP] in any specific intracellular compartment. Nevertheless, it is likely that intracellular free [ADP] for the entire functioning organ determined in this way is related to the [ADP] near the myofibril.
It has been suggested that glycolytic ATP plays a special role in diastolic relaxation by supporting the sarcoplasmic reticulum Ca2+-ATPase activity.32 Using 2DG perfusion as a strategy to increase free [ADP], we also perturbed glycolysis in these hearts. There is a possibility that loss of glycolytic ATP may be responsible for the diastolic dysfunction in the LVH hearts. Our study was designed to minimize this possibility. In hearts supplied with glucose as a sole substrate, glycolytic ATP contributes 3% to 4% of total ATP production, and this contribution is enhanced when high glucose concentration is used and insulin is included in the perfusate. With 5 mmol/L pyruvate supplied to the heart, the ATP synthesis via glycolysis was kept minimal even in the presence of 5 mmol/L glucose (<1% of total ATP production).12 Thus, hearts studied here are relatively independent of the confounding factor of glycolytic ATP. In support of this conclusion, perturbing glycolysis in the control hearts did not cause diastolic dysfunction. Our data do not allow us to rule out the possibility that hypertrophied hearts may be more dependent on glycolytic ATP for cross-bridge cycling and that a very small amount of glycolytic ATP (<100 μmol/L) is critical for diastolic relaxation in LVH hearts.
In summary, we found a close relationship between an increase in intracellular free [ADP] and impairment of diastolic relaxation in hearts with cardiac hypertrophy. Decreased energy reserve via the CK reaction may be an important mechanism that contributes to the impaired capacity to maintain a low free [ADP] in hypertrophied hearts.
Selected Abbreviations and Acronyms
|LVDP||=||LV developed pressure|
|LVEDP||=||LV end-diastolic pressure|
|LVH||=||LV hypertrophy (aortic-banded hearts)|
|LVSP||=||LV systolic pressure|
This study was supported by National Institutes of Health grants HL-44431 (to Dr Lorell), HL-49574 (to Dr Judith K. Gwathmey and Dr Ingwall), HL-52350 (to Dr Ingwall), and HL-52864 (to Dr Lorell). Dr Luigino Nascimben was supported by a Merck Sharp & Dohme Italia S.p.a. fellowship award. We would like to thank Drs Liang Zhao and Judith K. Gwathmey for helpful discussions of this study and Dr Ellen O. Weinberg for assistance with the animal model. The expert surgical assistance of Soeun Ngoy in the preparation of the aortic-banded rats and the skillful technical assistance of Ilana Reis are greatly appreciated.
- Received December 2, 1996.
- Revision received February 20, 1997.
- Accepted February 28, 1997.
- Copyright © 1997 by American Heart Association
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