© 1999 American Heart Association, Inc.
Original Contribution |
Adenylate Kinase–Catalyzed Phosphotransfer in the Myocardium
Increased Contribution in Heart Failure
From the Division of Cardiovascular Diseases, Mayo Clinic, Rochester, Minn. The current affiliation for Dr K.T. Vitkevicius is Kaunas Medical University, Kaunas, Lithiuania.
Correspondence to A. Terzic, Guggenheim-7, Mayo Clinic, Rochester, MN 55905. E-mail terzic.andre{at}mayo.edu
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Abstract |
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Abstract—Although the downregulation of creatine kinase activity has been associated with heart failure, creatine kinase–deficient transgenic hearts have a preserved contractile function. This suggests the existence of alternative phosphotransfer pathways in the myocardium, the identity of which is still unknown. In this study, we examined the contribution of adenylate kinase–catalyzed phosphotransfer to myocardial energetics. In the isolated mitochondria/actomyosin system, which possesses endogenous adenylate kinase activity in both compartments, substrates for adenylate kinase promoted the rate and amplitude of actomyosin contraction that was further enhanced by purified adenylate kinase. Inhibition of adenylate kinase activity diminished both actomyosin contraction and mitochondrial respiration, which indicated reduced energy flow between mitochondria and myofibrils. In intact myocardium, the net adenylate kinase–catalyzed phosphotransfer rate was 10% of the total ATP turnover rate as measured by 18O-phosphoryl labeling in conjunction with gas chromatography and mass spectrometry. In pacing-induced failing heart, adenylate kinase–catalyzed phosphotransfer increased by 134% and contributed 21% to the total ATP turnover. Concomitantly, the contribution by creatine kinase dropped from 89% in normal hearts to 40% in failing hearts. These phosphotransfer changes were associated with reduced levels of metabolically active ATP but maintained overall ATP turnover rate. Thus, this study provides evidence that adenylate kinase facilitates the transfer of high-energy phosphoryls and signal communication between mitochondria and actomyosin in cardiac muscle, with an increased contribution to cellular phosphotransfer in heart failure. This phosphotransfer function renders adenylate kinase an important component for optimal myocardial bioenergetics and a compensatory mechanism in response to impaired intracellular energy flux in the failing heart.
Key Words: heart failure • bioenergetics • adenylate kinase • creatine kinase • mitochondria • actomyosin
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Introduction |
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In cardiomyocytes, the generation of ATP primarily occurs in the mitochondria, which are separated from ATP-consuming sites in the myofibrils.1 To ensure communication between sites that generate and use ATP, it has been hypothesized that cells rely on phosphotransfer networks that facilitate the transfer and distribution of energy-rich phosphoryls between cellular compartments in a kinetically and thermodynamically efficient manner.2 3 4 5 6 7 Several studies have indicated that most phosphoryls are transferred through multiple creatine kinase–catalyzed phosphoryl exchanges that involve creatine phosphate and ATP.8 9 10 11
In fact, this enzyme is essential in supporting cardiac bioenergetics,5 7 10 12 and a decrease in energy reserve due to abnormalities in creatine kinase may contribute to heart failure.13 14 15 However, neither the suppression of creatine kinase activity7 nor the disruption of genes that encode the cytosolic and mitochondrial creatine kinase isoforms6 16 17 18 19 20 produce overt ventricular dysfunction. This suggests that alternative phosphotransfer routes may also support cardiac function. The identity of such phosphotransfer systems and their contribution to myocardial bioenergetics in the normal or failing heart remain unknown.
A candidate enzyme is adenylate kinase,4 21 22 23
which in skeletal muscle can transfer from 3% to 23% of the
high-energy phosphoryls, which depends on the functional load of the
muscle.8 22 24 This enzyme catalyzes the reversible
reaction 2ADP
ATP+AMP and may process metabolic signals
associated with ATP use.2 4 21 26 27 In this case,
adenylate kinase has been implicated in the regulation of
metabolically sensitive ion channels and
transporters.3 28 29 30 In addition, disruption of the
adenylate kinase gene impedes the export of ATP from the
mitochondria.31 Although in the heart, isoforms of this
enzyme are found in the mitochondria, cytosol, and
membranes,21 28 32 it is unknown whether adenylate
kinase contributes to the delivery of ATP to ATP-consuming sites.
Therefore, we examined the contribution of adenylate kinase to the transfer of energy-rich phosphoryls in an isolated mitochondrial/actomyosin system and in intact cardiac muscle. We report that adenylate kinase accelerates energy transfer and promotes communication between mitochondria and actomyosin, with an increased contribution to cellular phosphotransfer in the failing heart. This indicates a previously unrecognized function for this enzyme in cardiac muscle.
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Materials and Methods |
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Isolated Actomyosin/Mitochondrial System
Mitochondria and actomyosin were isolated from rabbit (Kaunas Medical Academy, Kaunas, Lithiuania) hearts.33 34 Mitochondria (at 0.08 mg of protein per mL) were coincubated with actomyosin (0.5 mg/mL) in (mmol/L): sucrose 300, Tris-HCl 10 (pH 7.4), KH2PO4 5, MgCl2 3,
-ketoglutarate 4, and
EGTA-Ca2+ 3. To maintain a steady flux of
high-energy phosphoryls between mitochondria and actomyosin, conditions
were chosen to match ATP consumption by actomyosin ATPases with ATP
production by mitochondria. The amount of actomyosin was
optimal for spectrophotometric recording of contraction, which
was based on changes in absorbance as an indicator of actin-myosin
interaction.35 Contraction was initiated by 100
µmol/L ADP or 10 µmol/L ATP and expressed as an increase in
absorbance at 540 nm.34 Mitochondrial respiration was
measured by O2 electrodes.33 The
respiratory control ratio of mitochondria-oxidizing NAD-dependent
substrates was 6.98±0.41 and 11.10±0.62 (n=10) with and without
Mg2+, respectively.
Heart Failure
To induce heart failure, dogs were paced at incremental rates
during 38 days.36 37 Ventricular pacing was
initiated at 180 bpm for 10 days. The rate was increased at 7-day
intervals to 200, 210, 220, and 240 bpm. This protocol results in
severe heart failure with systolic and diastolic
dysfunction; reduced ejection fraction, blood pressure, and cardiac
output; and elevation in left ventricular filling
pressure.36 37 The neurohumoral profile in this model
includes the activation of natriuretic peptides,
renin-angiotensin, and endothelin.36 37 From
normal and failing hearts, a segment of the left
ventricular wall was excised, washed, and placed in
ice-cold solution that contained 120 mmol/L NaCl and 20
mmol/L KCl. Samples were used immediately for
18O-phosphoryl labeling or stored at -80°C for
enzymatic analysis.
Enzyme Activity
Frozen samples were powdered in liquid N2
with mortar and pestle and extracted with 150 mmol/L NaCl, 60
mmol/L Tris-HCl (pH 7.5), 5 mmol/L EDTA, 0.2% Triton X-100,
1 mmol/L PMSF, 10 µg/mL leupeptin, and 1 µg/mL aprotinin.
Extracts were centrifuged (10 minutes, 10 000g,
4°C), and enzyme activities were measured with coupled enzyme assays
and with a Beckman DU 7400 spectrophotometer at 340
nm.8 38 Creatine kinase activity was measured in
100 mmol/L Tris-acetate (pH 7.5), 20 mmol/L glucose, 2
mmol/L EDTA, 10 mmol/L MgCl2, 2 mmol/L
dithiothreitol, 2 mmol/L NADP+, 2
mmol/L ADP, 5 mmol/L AMP, and 50 µmol/L
P1,
P2-diadenosine-5[p]pentaphosphate
(Ap5A), an adenylate kinase
inhibitor.39 This was supplemented with 4.5
U/mL hexokinase and 2 U/mL glucose-6-phosphate dehydrogenase, and the
reaction was started with 20 mmol/L creatine phosphate. Adenylate
kinase activity was measured in 100 mmol/L potassium acetate,
20 mmol/L HEPES (pH 7.5), 20 mmol/L glucose, 4 mmol/L
MgCl2, 2 mmol/L NADP+,
1 mmol/L EDTA, 1 mmol/L dithiothreitol, 4.5 U/mL hexokinase,
and 2 U/mL glucose-6-phosphate dehydrogenase, and the reaction was
initiated with 2 mmol/L ADP (purified by HPLC).
Phosphoryl Flux in Intact Heart Muscle
ATP turnover and phosphoryl flux through adenylate and
creatine kinases were measured in intact cardiac muscle with the
18O-phosphoryl–labeling technique.8
This procedure is based on the incorporation of 1
18O atom in inorganic phosphate with each act of
ATP hydrolysis and the distribution of
18O-labeled phosphoryls among high-energy
phosphoryl-carrying molecules that depend on the flux via specific
phosphotransfer reactions.24 25 Therefore, a method to
quantify flux through individual enzymatic pathways is to monitor the
kinetics of 18O-phosphoryl–oxygen
exchange.22 Ventricular slices (1- to 2-mm
thick) were washed 3 times, preincubated (15 minutes, 37°C) in
oxygenated Krebs-Henseleit buffer (118 mmol/L NaCl;
4.7 mmol/L KCl; 1.8 mmol/L CaCl2;
1.2 mmol/L MgCl2; 0.5 mmol/L EDTA;
25 mmol/L NaHCO3; 11 mmol/L glucose;
and 10 U/L insulin, pH 7.45), and transferred into 95%
O2/5% CO2 saturated
buffer, in which 20% to 30% of water was replaced with
18O-containing water. After 1, 2, 4, 6, and 9
minutes of incubation, slices were freeze-clamped, pulverized in mortar
with liquid N2, and extracted.8
Cellular ATP, ADP, inorganic phosphate, and creatine phosphate were
purified and quantified with HPLC.30 The 
-phosphoryl of
ATP was transferred to glycerol by glycerokinase, and the
ß-phosphoryls of ATP and ADP were transferred to glycerol by a
combined catalytic action of adenylate kinase and
glycerokinase. The phosphoryl of creatine phosphate was transferred to
-ATP by creatine kinase and then to glycerol with glycerokinase.
Samples that contained phosphoryls of -ATP, ß-ATP, ß-ADP,
inorganic phosphate, and creatine phosphate, as glycerol 3-phosphate,
were converted to respective trimethylsilyl derivatives. The
18O-enrichment of phosphoryls in glycerol
3-phosphates was determined with a Hewlett-Packard 5980B gas
chromatograph-mass spectrometer operated in the select
ion-monitoring mode. Mass ions (m/z) of 357, 359, 361, and 363 that
corresponded to phosphoryl species of
18O0,
18O1,
18O2, and
18O3 were monitored. The
trimethylsilyl derivative of orthophosphate yielded mass ions (m/z) of
299, 301, 303, 305, and 307 that corresponded to species of
orthophosphate that contained 0, 1, 2, 3, and 4 atoms of
18O. The percentage of nucleotide
phosphoryl oxygens replaced by 18O was calculated
as
[%18O1+2(%18O2)+3(%18O3)]/[3(%18OxH2O)].30
Total cellular ATP turnover was estimated from the sum of the total
number of 18O atoms that appeared in
phosphoryl-containing metabolites and orthophosphate.8 24
The net rate of adenylate kinase–catalyzed phosphotransfer was
estimated from the initial rate of appearance of
18O-containing ß-phosphoryls in ADP and
ATP.8 The net rate of creatine kinase–catalyzed
phosphoryl transfer was determined from the initial rate of appearance
of creatine phosphate species with 18O-labeled
phosphoryls.8 24
Statistical Analysis
Data are expressed as mean±SEM. Student t test for
unpaired samples was used for statistical analysis, and a
difference at P<0.05 was considered significant.
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Results |
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Adenylate Kinase–Mediated Communication Between Isolated Actomyosin and Mitochondria
Isolated mitochondrial (n=5) and actomyosin (n=10) preparations possessed endogenous adenylate kinase activity of 469±49 and 83±5 nmol ATP · min-1 · mg-1 protein, respectively, and were coincubated at protein concentrations in which the mitochondrial/myofibrillar ratio of adenylate kinase activity was
1:1. When ADP, a substrate of adenylate kinase and
mitochondria, was added to the mitochondrial/actomyosin system,
actomyosin contraction was promoted, as observed by increased
absorbance of the actomyosin complex (Figure 1A
, curve 1). Treatment with
Ap5A39 slowed the development and
decreased the amplitude of actomyosin contraction (Figure 1A).
The rate and amplitude of contraction were 150±25 absorbance units
(AU)-3 · min-1 and
0.089±0.004 AU in the absence versus 21±3
AU-3 · min-1 and
0.015±0.001 AU in the presence of Ap5A (n=10;
Figure 1B). Values measured in the presence of
Ap5A were significantly lower than those obtained
in the absence of the adenylate kinase inhibitor
(P<0.001). The addition of exogenous, purified,
adenylate kinase (7 U/mL) significantly (P<0.001)
accelerated the rate (325±49 AU-3 ·
min-1) and increased the amplitude (0.138±0.006
AU) of contraction (Figure 1A and 1B); both effects were
inhibited by Ap5A (not shown). Thus,
adenylate kinase activity promotes actomyosin contraction,
which could be due to local regeneration of ATP and/or increased
transfer of ATP from mitochondria to myosin ATPases.
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To distinguish between these 2 interrelated functions of
adenylate kinase, the local regeneration of ATP versus the
spatially-directed transfer of nucleotides between
mitochondria and actomyosin,4 23 we examined whether
adenylate kinase activity promotes contraction under conditions
in which ATP regeneration is minimal and diffusional limitations exist
for adenine nucleotide exchange between mitochondria and
actomyosin. In the absence of mitochondria, when actomyosin was induced
to contract by the addition a low concentration of ATP (10
µmol/L), the rate and amplitude of contraction were 167±6
AU-3 · min-1 and
0.051±0.003 AU (n=5; Figure 2A). In the
continued absence of mitochondria and presence of
Ap5A, the rate and amplitude of contraction were
161±6 AU-3 ·
min-1 and 0.043±0.002 AU (n=8; Figure 2A). Values obtained in the absence and presence of the
adenylate kinase inhibitor were not significantly
different (P>0.05), which suggested that in the absence of
mitochondria and at a low concentration of ATP, the ATP-regenerating
function of adenylate kinase is minimal. Additional
mitochondria in the continued presence of Ap5A
accelerated the rate of contraction to 216±5
AU-3 · min-1 and
increased the amplitude of contraction to 0.065±0.002 AU
(P<0.05, n=8; Figure 2), which suggested that to
some extent energy can be transferred by simple diffusion or other
phosphotransfer systems in the absence of adenylate
kinase–catalyzed phosphotransfer. In the presence of mitochondria but
absence of Ap5A, the rate and amplitude of
contraction were 282±9 AU-3 ·
min-1 and 0.086±0.003 AU (n=8); these values
are significantly higher than those measured in the presence of
Ap5A (P<0.001; Figure 2A).
Thus, in the reconstituted system, adenylate kinase promotes
the delivery of ATP from mitochondria to actomyosin ATPases even in the
absence of a major effect on ATP regeneration.
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To assess whether adenylate kinase contributes to feedback communication between actomyosin and mitochondria, mitochondrial respiration was measured in the absence and presence of adenylate kinase inhibition. In the absence of Ap5A, mitochondria respired at 203±10 nanoatoms (natom) O2 · min-1 · mg-1 protein (n=5). This value is between values that correspond to state 4 (49±3 natom O2 · min-1 · mg-1 protein) and state 3 (351±27 natom O2 · min-1 · mg-1 protein) of the mitochondrial respiratory rate determined in the absence and presence of ADP (200 µmol/L), respectively. This indicates that mitochondria participate in the regeneration of ATP consumed by myosin ATPases. The addition of Ap5A (50 µmol/L) reduced mitochondrial respiration to 172±10 natom O2 · min-1 · mg-1 protein (n=5), which was significantly lower than the value obtained in the absence of the adenylate kinase inhibitor (P<0.001). Thus, inhibition of adenylate kinase compromised communication between actomyosin and mitochondria.
Adenylate Kinase–Catalyzed Phosphotransfer in Intact
Myocardium
The contribution of adenylate kinase to cellular
phosphotransfer was assessed in myocardial samples with
18O-phosphoryl labeling. The kinetics of
18O-labeled phosphoryl appearance in -ATP
reflects cellular ATP turnover rate.24 The total ATP
turnover rate, which is determined from the sum of
18O atoms inserted into phosphoryl-containing
cellular metabolites, was 37.5±1.4 nmol ATP ·
min-1 · mg-1
protein (n=5; Figure 3A and 3B).
Adenylate kinase catalyzes ß-ATP
18O-labeling.8 The net
adenylate kinase–catalyzed phosphotransfer rate in the
direction of ADP formation, was 3.72±0.3 nmol ADP ·
min-1 · mg-1
protein (n=5; Figure 3A and 3B). This value corresponds to
10% of the total ATP turnover rate. The rate of
18O-labeling of creatine phosphate reflects
creatine kinase–catalyzed phosphotransfer.8 The net
creatine phosphate–catalyzed phosphotransfer rate was 32.4±1.6 nmol
CrP · min-1 ·
mg-1 protein (n=5) or 89% of the total ATP
turnover rate (Figure 3A and 3B). Thus in intact
myocardium, adenylate kinase can transfer
high-energy phosphoryls that contribute to cellular
phosphotransfer.
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Increased Contribution of Adenylate Kinase in Heart
Failure
The kinetics of 18O-labeling of phosphoryls
in -ATP, creatine phosphate, and ß-ATP in failing hearts are
presented in Figure 3C
. The overall ATP turnover rate
(42.1±1.5 nmol ATP · min-1 ·
mg-1 protein, n=6) was essentially unchanged
(P>0.05) when compared with controls (Figure 3A).
Although the initial rate of 18O-labeled
phosphoryl appearance in -ATP was similar in the 2 groups,
subsequent 18O incorporation was reduced in the
failing myocardium and approached saturation faster, which
suggested that in heart failure, a smaller pool of ATP is involved in
energy turnover (Figure 3A through 3C). Thus at 6 minutes of
18O-labeling, the pool size of labeled -ATP
was lower by 20% (P<0.05) in the failing
myocardium compared with normal myocardium
(Figure 3A through 3C). The failing heart also displayed a
marginal decrease in ATP levels to 25.7±2.3 nmol ·
mg-1 protein (n=6) from 29.2±2.5 nmol ·
mg-1 protein (n=5) found in normal hearts
(P>0.05). There was a reduction (by 52%) in creatine
kinase-catalyzed 18O-labeling of creatine
phosphate to 17.4±0.7 nmol · mg-1
protein · min-1 (n=6;
P<0.001; Figure 3D). In failing hearts, the
contribution of creatine kinase to the overall ATP turnover rate
decreased to 40%. But, adenylate kinase–catalyzed ß-ATP
18O-labeling increased. The rate of
adenylate kinase–catalyzed phosphotransfer increased to
8.71±0.4 nmol · mg-1 protein ·
min-1 (n=6) or 134% compared with controls
(P<0.001). In the failing myocardium, the
contribution of adenylate kinase to the overall ATP turnover
doubled and was 21% of the total ATP turnover rate.
To assess whether changes in phosphoryl fluxes are related to altered enzyme activities, specific activities of adenylate and creatine kinase were measured. In control hearts (n=6), the activities of adenylate and creatine kinases were 537±18 and 2551±64 nmol · min-1 · mg-1 protein, respectively. In extracts from failing hearts (n=8), specific activities of adenylate and creatine kinases were 422±16 and 1791±42 nmol · min-1 · mg-1 protein, respectively. These are significant (P<0.001) decreases in specific activities when compared with controls: 21% for adenylate and 30% for creatine kinase. Thus, the increase in phosphoryl flux through adenylate kinase in failing hearts occurs despite a reduction in the total activity of this enzyme.
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Discussion |
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In the heart, adenylate kinase has been implicated in the control of oxidative phosphorylation,26 33 40 export of high-energy phosphoryls from mitochondria,21 and regulation of ATP-sensitive K+ channels.3 28 41 In this study, we show that adenylate kinase also facilitates the transfer of high-energy phosphoryls and signal communication between mitochondria and actomyosin. This was demonstrated in an isolated actomyosin/mitochondria system and in intact myocardium. The contribution of adenylate kinase to the overall cellular ATP metabolic flux significantly increased during heart failure when creatine kinase–dependent phosphotransfer was compromised. Thus, this study establishes the role for adenylate kinase in supporting myocardial energy flux.
In the reconstituted actomyosin/mitochondrial system devoid of other ATP-regenerating and ATP-consuming cellular components,34 adenylate kinase–catalyzed phosphotransfer, which can occur in mitochondria and myofibrils,33 42 accelerated the rate and increased the amplitude of contraction. This effect was suppressed by Ap5A,39 which has no direct effect on myosin ATPases or mitochondria.43 44 A dependence of contraction on adenylate kinase activity could be due to local regeneration of ATP and/or transfer of nucleotides between mitochondria and actomyosin.4 25 When conditions were able to minimize the ATP-regenerating function of adenylate kinase at low concentrations of ADP or ATP, the effect of adenylate kinase–catalyzed phosphotransfer required both actomyosin and mitochondria. In the absence of mitochondria, the ATP-regenerating function of adenylate kinase is limited by the accumulation of AMP.33 40 In the presence of mitochondria, AMP is rephosphorylated to ATP and transferred back to the myofibrils, which allows for continuous phosphoryl flux.4 Yet, in the absence of adenylate kinase activity, the exchange of energy-rich nucleotides by simple diffusion is insufficient to sustain a maximal rate of contraction. But, combined actomyosin/mitochondrial adenylate kinase activities promoted actomyosin contraction and mitochondrial respiration, which indicated an adenylate kinase–dependent flow of high-energy phosphoryls from mitochondria to actomyosin. These findings fulfill criteria for adenylate kinase–catalyzed phosphotransfer to process cellular signals associated with ATP production and use in cardiomyocytes, as proposed previously for noncardiac tissues.3 4 8 22 30
In intact cardiac muscle, the total ATP turnover rate (37.5 nmol ATP · min-1 · mg-1 protein) was obtained directly from 18O measurements and was within the range (27 to 54 nmol ATP · min-1 · mg-1 protein) of that calculated24 from O2 consumption rates in resting dog myocardium.45 46 The net adenylate kinase–catalyzed phosphotransfer rate accounted for 10% of the total ATP turnover rate, compared with 3% to 5% in resting skeletal muscle,8 22 which suggested possible tissue-specific differences in the contribution of adenylate kinase to cellular phosphotransfer. In this experiment, creatine kinase phosphotransfer contributed 89% to the total ATP turnover rate. Thus, in the heart, the majority of high-energy phosphoryls are transferred through combined actions of creatine and adenylate kinases.
In the failing myocardium, the overall ATP turnover rate
was similar to that in the normal heart, which supported the notion
that ATP levels and ATPase activity may not change with the progression
of disease.1 In fact, we did not observe a significant
decrease in the ATP concentration in the failing
myocardium. However, changes in the kinetics of -ATP
18O-labeling suggest that in heart failure a
smaller pool of ATP is actively involved in cellular energetics. This
can result from altered compartmentation and/or deficient
phosphotransfer of adenine nucleotides.7 10 11
Present data show a reduction (by 52%) in creatine
kinase–catalyzed phosphotransfer in pacing-induced heart failure and a
decreased (from 89% to 40%) contribution of creatine kinase to the
overall ATP turnover. This correlates with previous studies that have,
in other models of heart failure, also implicated a deficiency in
creatine kinase–catalyzed energy transfer.15 47 The new
information provided by this study relates to the significance of
adenylate kinase–catalyzed phosphotransfer that increased by
134% in heart failure. Consequently, the contribution of
adenylate kinase to total ATP turnover rate doubled to 21%.
Such increase in adenylate kinase–catalyzed phosphotransfer
could support cellular energetics of a failing heart.
Compensation provided by adenylate kinase was only partial. The
sum of phosphotransfers mediated through creatine and adenylate
kinases contributed to 100% of the total ATP turnover rate in
normal hearts but only 60% to 65% in failing hearts, which left a
35% to 40% phosphotransfer deficit. This term refers to a deficit in
enzyme-mediated phosphotransfer in which remaining phosphoryls are
delivered through a less-efficient mechanism, such as simple diffusion
and/or the glycolytic pathway.3 4 Although in normal
muscle an acute suppression of creatine kinase activity is well
compensated through increased adenylate kinase
phosphotransfer,8 the compensatory potential of
adenylate kinase in diseased heart appears to be reduced.
Previously, in ischemia-injured myocardium, a
diminished activity of adenylate kinase has been reported to
reduce the export of high-energy phosphoryls from
mitochondria.48 The specific activity of adenylate
kinase reflects the flux capacity of this enzyme. Therefore, a drop in
flux capacity, as observed here in the failing myocardium,
may limit the compensatory potential of adenylate
kinase–dependent phosphotransfer. This could be due to a decrease in
adenylate kinase expression and/or altered enzyme regulation.
Because our preliminary results, which were performed with Western blot
analysis to detect adenylate kinase level in
ventricular samples, do not indicate a significant decrease
in protein levels of adenylate kinase in heart failure, other
mechanisms should be considered including changes in cardiac levels of
Ap5A that may occur under metabolic
stress.49
In summary, the current study, performed in the isolated
mitochondria/actomyosin system and intact ventricular
tissue, identifies a role for adenylate kinase in the transfer
of energy and feedback communication between mitochondria and
myofibrils in the heart. Although experimental systems permit the
demonstration of phosphotransfer dynamics that are independent from
variations due to muscle contraction, the functional integrity of
cardiac muscle is only partially preserved, which warrants additional
studies to establish the full role of adenylate kinase in a
beating heart. A coordinated action of mitochondrial and cytosolic
isoforms of adenylate kinase, which are encoded by separate
genes and directed to different subcellular
compartments,32 would provide a mechanism to transfer 2
high-energy phosphoryls within the ATP molecule (Figure 4). The exclusive property of
adenylate kinase to transfer the energy of the ß-phosphoryl
of ATP, which is energetically equivalent to the commonly employed
-phosphoryl, doubles the energetic potential of the ATP molecule and
halves the diffusional resistance of the cytosol for energy
transfer.4 50 Such phosphotransfer function renders
adenylate kinase an important component for optimal myocardial
bioenergetics. In particular, adenylate kinase could serve a
compensatory role in heart failure. Thus, the promotion of
adenylate kinase–mediated phosphotransfer could provide a
novel strategy targeted at improving the energetic status of the
failing heart.
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Acknowledgments |
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This work was supported by grants from the NIH (HL-07111 and HL-36634), American Heart Association, Miami Heart Research Institute, and the Bruce and Ruth Rappaport Program in Vascular Biology and Gene Delivery.
Received November 20, 1998; accepted March 12, 1999.
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