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Energetic Crosstalk Between Organelles
Architectural Integration of Energy Production and Utilization
Allen Kaasik, Vladimir Veksler, Ernest Boehm, Marta Novotova, Ave Minajeva, Renée Ventura-Clapier
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Abstract—Cells with high and fluctuating energy demands such as cardiomyocytes need efficient systems to link energy
production to energy utilization. This is achieved in part by compartmentalized energy transfer enzymes such as creatine
kinase (CK). However, hearts from CK-deficient mice develop normal cardiac function under conditions of moderate
workload. We have therefore investigated whether a direct functional interplay exists between mitochondria and
sarcoplasmic reticulum or between mitochondria and myofilaments in cardiac cells that catalyzes direct energy and
signal channeling between organelles. We used the selective permeabilization of sarcolemmal membranes with saponin
to study the functional interactions between organelles within the cellular architecture. We measured contractile kinetics,
oxygen consumption, and caffeine-induced tension transients. The results show that in hearts of normal mice, ATP
produced by mitochondria (supplied with substrates, oxygen, and adenine nucleotides) was able to sustain calcium
uptake and contractile speed. Moreover, direct mitochondrially supplied ATP was nearly as effective as CK-supplied
ATP and much more effective than externally supplied ATP, suggesting that a direct ATP/ADP channeling exists
between the sites of energy production (mitochondria) and energy utilization (sarcoplasmic reticulum and myofilaments). On the other hand, in cardiac cells of mice deficient in mitochondrial and cytosolic CK, marked cytoarchitectural
modifications were observed, and direct adenine nucleotide channeling between mitochondria and organelles was still
effective for sarcoplasmic reticulum and myofilaments. Such direct crosstalk between organelles may explain the
preserved cardiac function of CK-deficient mice under moderate workloads. (Circ Res. 2001;89:153-159.)
Key Words: mitochondria 䡲 sarcoplasmic reticulum 䡲 myofibrils 䡲 creatine kinase 䡲 knockout mice
D
ifferentiation and maturation of adult mammalian muscle cells lead to complex specialization and organization. In cardiac cells, specialized cellular functions are highly
organized within structural and functional compartments.
Energy-consuming processes are localized to the sarcoplasmic reticulum (SR) and myofibrillar compartments, while
energy production occurs mainly within mitochondria. Muscle cells contain complex and specialized energy transfer
systems, which efficiently link energy production and utilization. One such system is the family of creatine kinase (CK)
isoenzymes that catalyze the reversible transfer of a phosphate moiety between creatine (Cr) and ATP. The mitochondrial sarcomeric isoenzyme (mi-CK) is bound to the outer
surface of the inner mitochondrial membrane so that ATP
generated by oxidative phosphorylation is transphosphorylated to phosphocreatine (PCr).1–3 On the other hand, the
cytosolic isoenzyme (MM-CK) that is structurally associated
with myofibrils and SR membranes can use PCr to rephosphorylate all of the ADP produced by the ATPases and thus
provide enough energy for normal contractile kinetics or SR
calcium uptake.4 –7
Recent studies have revealed that mice lacking one or both
of the MM-CK and mi-CK isoforms (CK⫺/⫺) are viable and
develop nearly normal cardiac function under the conditions
of moderate workload.8 –11 This suggests that other mechanisms may ensure efficient energy transfer and signal transduction between sites of energy production and energy
utilization. Indeed, increases in mitochondrial volume and
cytoarchitectural rearrangements have been observed in
CK⫺/⫺ mice10 suggesting adaptational mechanisms to CK
deficiency. One possibility might be that a direct functional
interplay between subcellular organelles exists that catalyzes
direct energy and signal channeling between mitochondria
and the SR on the one hand and between mitochondria and
myofilaments on the other. Indeed, mitochondria appear to be
clustered at sites of high ATP demand and are organized into
highly ordered elongated bundles, wrapped around the myofibrils and in contact with the SR.12 Structural contacts
between the SR and mitochondria have been revealed by
electron microscopy,13 and compelling evidence points to a
coordination between these organelles at the level of calcium
homeostasis14 –16 and regulation of ATP production.17 Previous studies have suggested a possible direct functional
interaction between these ATP-producing and -consuming
intracellular organelles at the level of energy transfer.18 –20
Original received November 27, 2000; revision received May 23, 2001; accepted May 23, 2001.
From the Cardiologie Cellulaire et Moléculaire U-446 INSERM (A.K., V.V., E.B., A.M., R.V.-C.), Université Paris-Sud, Châtenay-Malabry, France;
Molecular Physiology and Genetics (M.N.), Slovak Academy of Sciences, Bratislava, Slovak Republic. Present address for A.K. is Department of
Pharmacology, University of Tartu, Tartu, Estonia; present address for E.B. is Wellcome Trust Centre for Human Genetics, Oxford, UK.
Correspondence to Renée Ventura-Clapier, U-446 INSERM, Faculté de Pharmacie, 92 296 Châtenay-Malabry, France. E-mail [email protected]
© 2001 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
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Functional studies using skinned fibers provide a unique
mean to investigate these possible interactions. Indeed, the
use of specific membrane permeabilization with detergents
allows for the study of organelle function while maintaining
the cellular architecture and controlling the intracellular
milieu. This experimental approach is a valuable tool for
studying mitochondrial function and regulation in situ and
can demonstrate the functional coupling between bound
MM-CK and cardiac sarcoplasmic reticulum (SERCATPase) or myofibrillar ATPase.4,6,21 In the present study,
using the selective permeabilization of cardiac sarcolemmal
membranes with saponin, we have investigated (1) whether
direct ATP supply by mitochondria can provide energy to the
SERC-ATPase for calcium uptake or to myosin-ATPase for
contraction, (2) whether ATP supplied directly by mitochondria is as effective as ATP supplied by bound CK or as ATP
supplied from the surrounding medium, and (3) how ATP is
supplied to energy-utilizing organelles in cardiac cells of
mice lacking sarcomeric mitochondrial and cytosolic CKs
(CK⫺/⫺ mice).
Materials and Methods
Preparation of Skinned Fibers and Solutions
Procedures involved in the generation and genotyping of
mitochondrial/MM-CK null mice have been described in detail
elsewhere.10 Three-month-old male control C57BL/6 and CK⫺/⫺
mice were anesthetized with an intraperitoneal injection of sodium
thiopental according to the recommendations of the Institutional
Animal Care Committee (INSERM, Paris, France). The hearts were
removed and rinsed in an ice-cold Ca2⫹-free Krebs solution equilibrated with 95%O2/5% CO2. Fibers (diameter 150 to 250 ␮m) were
dissected from left ventricular papillary muscles. Specific permeabilization of sarcolemma was obtained by incubating the fibers for 30
minutes in relaxing solution (basic solution at pCa 9, see below)
containing additionally 50 ␮g/mL saponin in the presence of 5
␮g/mL leupeptin at 4°C. After skinning, fibers were kept in the
relaxing solution at 4°C until further use. Solutions for permeabilized
fibers were calculated using the computer program of Fabiato.22 All
solutions were prepared using the basic solution. This solution
contained (in mmol/L) ethylene glycol-bis(␤-aminoethyl ether)
N,N,N⬘,N⬘-tetra-acetic acid 10 (EGTA; except for prerelease and
release solutions, 0.2), N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid 60 (BES, pH 7.1), free Mg2⫹ 1, taurine 20, glutamic acid
5, malic acid 2, K2HPO4 3, dithiothreitol 0.5, P1,P5 diadenosine
pentaphosphate 0.040 (to inhibit adenylate kinase activity), and
MgATP 3.16 (except otherwise stated); ionic strength was adjusted
to 160 mmol/L with potassium methanesulfonate. Desired pCa was
obtained by varying CaK2EGTA/K2EGTA ratio.
Electron Microscopy Study
Samples of the left ventricles, taken from 5 hearts of wild-type or
CK⫺/⫺ mice, were washed with Ca2⫹-free Krebs solution for 10
minutes and fixed with 2% glutaraldehyde. After fixation, tissue
samples were postfixed with 1% osmium tetroxide, contrasted with
1% uranyl acetate in ethanol, dehydrated, and embedded in Durcupan (Fluka Chemie AG). Ultrathin longitudinal sections were stained
with lead citrate and studied using a JEM 1200 (JEOL) electron
microscope.
Estimation of SR Ca2ⴙ Uptake
The experimental protocol used in this study was a modified version
of that described by Minajeva et al4 (for more detail, see the online
data supplement available at http://www.circresaha.org). After emptying the SR by a brief application of caffeine (5 mmol/L), SR
loading was carried out in solutions with different ATP sources at
pCa 6.5. These solutions contained in addition to basic solution
(in mmol/L) ADP 1, instead of ATP for ADP⫹MITO solution; azide
2, to inhibit mitochondria for ATP solution or no azide for MITO
solution; PCr 12 and azide 2, for ATP⫹PCr solution; or no azide for
ATP⫹PCr⫹MITO solution. In some experiments, mitochondrial
substrates (glutamate and malate) were omitted or oligomycin (40
␮mol/L), an inhibitor of mitochondrial ATPase activity, was added.
Calcium release was induced by 5 mmol/L caffeine in the presence
of PCr and ATP for control mice or with ATP alone when comparing
control and CK⫺/⫺ mice (see below), to ensure comparable conditions of myofilament activation. Tension at peak and tension-time
integral were measured and analyzed as previously described.4
Myofibrillar Function
Myofibrillar crossbridge cycling rate, which is the functional counterpart of actomyosin ATPase activity, was estimated by the quick
length-change technique as previously described23 (for more detail,
see online data supplement).
Oxygen Consumption and
Biochemical Determinations
Respiratory rates were determined using a Clark electrode (Strathkelvin Instruments) as described previously24 (for more detail, see
online data supplement). For estimating the competition between
mitochondria and CK for ATP supply to ATPases, fibers (⬇0.35 mg
of dry weight) were transferred into 1 mL oxygraphic cell– containing basic solution at pCa 6.5 and the respiratory rate was determined.
Five minutes later, 12 mmol/L PCr was added to the chamber and
oxygen measurements continued for an additional 5 minutes. Thereafter, a mixture of atractyloside (20 ␮mol/L) and oligomycin (20
␮mol/L) was added to quickly stop mitochondrial respiration and
measurements continued for an additional 5 minutes. During all
these steps, samples from the respiration media were collected and
the Cr concentration was determined.
Statistical Analysis
Values are expressed as mean⫾SE. A Student’s t test was used to
determine the statistical difference of means between control and
CK⫺/⫺ groups. Within a group, statistical significance of differences
between the averages was estimated by a repeated-measures of
ANOVA using Dunnett’s post hoc test.
An expanded Materials and Methods section can be found in the
online data supplement available at http://www.circresaha.org.
Results
Mitochondria-Supported SR Ca2ⴙ Load
We previously reported that in saponin-permeabilized fibers,
the SR could be loaded much more efficiently with PCr and
ATP compared with ATP alone. We concluded that CK
bound to the SR membrane was able to provide ATP and
withdraw ADP close to the Ca2⫹-ATPase-driven pump.4,25
We wondered whether a functional compartmentation can
occur in normal hearts between the SR and mitochondria, two
organelles known to establish physical contacts. Fibers were
incubated in a solution containing mitochondrial substrates
and ADP (ADP⫹MITO solution) at pCa 6.5 for 5 minutes, to
load the SR at the expense of mitochondrially produced
energy. When sequestered calcium was released with caffeine, a tension transient could be elicited, showing that SR
has been effectively loaded with calcium (Figure 1, left). This
uptake was time-dependent as it increased with longer incubation periods (results not shown). Sodium azide, an inhibitor
of mitochondrial respiration, completely abolished mitochondrially supported SR loading. Thus, mitochondrially produced energy could time dependently support SR Ca2⫹
Kaasik et al
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155
Figure 1. Evidence for direct channeling of adenine nucleotides
between mitochondria and SERC-ATPase. Superimposed tension transients elicited by 5 mmol/L caffeine after 5 minutes of
SR loading with or without 2 mmol/L azide to inhibit mitochondria, in the presence of 1 mmol/L ADP (ADP) or 3.16 mmol/L
ATP (ATP).
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loading. However, these experiments were unable to answer
the question of the efficacy of the mitochondrially supported
SR load, because high ADP concentrations are known to
inhibit or reverse the SR calcium pump. We thus replaced
external ADP with ATP in the loading solutions. Under these
conditions, mitochondria can only use the ADP coming from
the hydrolysis of ATP catalyzed by the cellular ATPases.1 SR
load was up to 4-fold more effective when ATP was used
instead of ADP (Figure 1, right). Moreover, when mitochondria were blocked with 2 mmol/L sodium azide, only 5% of
the response remained, suggesting that most of the ATP used
was of mitochondrial origin and not from external ATP.
Thus, in normal hearts, mitochondrially produced ATP appeared far more effective in supporting SR calcium load than
exogenous ATP, showing that mitochondria can effectively
maintain high ATP/ADP ratio near the SR calcium pump.
Our next step was to compare the efficacy of mitochondrially produced ATP with the efficacy of ATP regenerated by
bound CK. For this, we compared the SR calcium release
after 5 minutes of loading at pCa 6.5 supported either (1) by
external ATP, (2) by external ATP and mitochondria, (3) by
external ATP and PCr, or (4) by external ATP, PCr, and
mitochondria. Either in the presence of active mitochondria
or PCr, or both, the SR load was significantly higher than it
was with external ATP alone (Figure 2). We checked whether
azide could induce an increase in mitochondrial ATPase
activity, leading to local ATP depletion. However, when
mitochondria were blocked by omitting substrates, or by
azide, or by azide with oligomycin, an inhibitor of mitochondrial ATPase activity, tension transients were much lower
than when SR was loaded with mitochondria (Figure 3A). In
the following experiments, azide was preferred because of
simplicity of use and high reversibility, which allowed the
loading conditions to be randomized. Averaged peak transients and tension-time integrals were much lower when
external ATP was supplied than when ATP was produced by
either CK or mitochondria (see online data supplement
available at http://www.circresaha.org). Normalizing tensiontime integrals to the ATP⫹MITO condition for each fiber
showed that the ATP-supported load was almost 30 times less
effective than other loading conditions (Figure 3C). As in
ATP⫹MITO, the tension-time integral increased linearly for
up to 15 minutes of loading (results not shown); SR was not
saturated at 5 minutes of loading for any conditions. Thus,
mitochondria can support SR calcium load almost as effectively as the PCr/CK system. Moreover, each loading condi-
Figure 2. Comparison of various sources of ATP supply to
SERC-ATPase in cardiac fibers. Tension transients elicited by
5 mmol/L caffeine after 5 minutes of SR loading with
3.16 mmol/L ATP⫹2 mmol/L azide (ATP), 3.16 mmol/L ATP
(ATP⫹MITO), or 3.16 mmol/L ATP⫹12 mmol/L PCr in the presence (ATP⫹PCr) or absence (ATP⫹MITO⫹PCr) of 2 mmol/L
azide to inhibit mitochondria. The routes of ADP and ATP are
indicated for each experimental condition (arrows).
tion seems to be maximally efficient, because loading in
ATP⫹PCr⫹MITO solution was not significantly higher than
in ATP⫹MITO.
Mitochondria-Supported Myosin ATPase Activity
In the next experiments, we checked whether mitochondrially
supplied ATP could also be effectively used for myofibrillar
function. It has been previously shown that bound CK is
necessary for optimal myosin ATPase activity and crossbridge cycling.26 Functional activity of myofibrils was estimated by measuring the rate constant of tension changes after
quick changes in length33 under the different ATP supply
conditions as used for the SR. Figure 3B shows that whatever
the conditions, when mitochondria were inhibited, crossbridge cycling rate was much lower than when ATP was
produced by mitochondria, showing again that the low
efficacy of external ATP was not due to activation of
mitochondrial ATPase. Results with different loading conditions are presented in Figure 3D as relative values (absolute
values provided in the online data supplement). The rate
constant increased significantly in the presence of active
mitochondria or PCr when compared with external ATP only.
In the presence of active mitochondria and PCr, the rate
constant was even higher than with active mitochondria
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Mitochondrial Respiration of Saponin-Permeabilized Ventricular
Fibers From Control and CKⴚ/ⴚ Mice
Control
V0, ␮mol O2 䡠 min⫺1 䡠 g dry weight⫺1
Vmax, ␮mol O2 䡠 min⫺1 䡠 g dry weight⫺1
Acceptor control ratio
CK⫺/⫺
5.7⫾1.0
5.7⫾0.3
22.4⫾3.0
23.0⫾1.6
4.3⫾0.4
4.0⫾0.3
483⫾84
185⫾22*
45⫾6†
223⫾26‡
11.4⫾2.0
0.9⫾0.2‡
Km for ADP, ␮mol/L
Without creatine
With creatine
Creatine kinase efficacy
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Figure 3. A, Tension-time integrals of tension transients
obtained after loading the SR without mitochondrial substrates
(w/o subs.), with substrates (MITO), after addition of azide to
inhibit mitochondria (⫹2 mmol/L azide), and further addition of
oligomycin (⫹40 ␮mol/L), an inhibitor of mitochondrial ATPase
activity. B, Crossbridge cycling rate measured in same conditions as in panel A. C, Tension-time integrals after loading the
SR with ATP alone (azide), MITO, PCr, or MITO⫹PCr. D, Rate
constants of crossbridge cycling of fibers in same conditions as
in panel C. Values are normalized to MITO load. *P⬍0.05,
repeated-measures ANOVA followed by Dunnett’s test. Results
are mean of 4 to 7 experiments.
alone. The results demonstrate that in myofibrils, as in SR of
normal cardiac cells, mitochondria can favor the ATP/ADP
compartmentation nearly as effectively as PCr.
Competition Between Mitochondria and CK
From the above experiments, it was clear that mitochondrially
produced energy alone was sufficient to support SR Ca2⫹ load
and myosin-ATPase activity to almost the same extent as
bound CK. However, it remained unclear which mechanism
of ATP regeneration dominates when both mechanisms are
working together. We therefore simultaneously estimated the
activity of the CK reaction by measuring Cr formation and
the mitochondrial ATP synthesis rate by measuring ADPdependent oxygen consumption (Figure 4). When both CK
and mitochondria were working, the rate of Cr formation
amounted to 53⫾3 nmol Cr · min⫺1 · mg dry weight⫺1
Figure 4. Model of ATP supply in cardiac fibers. Relative contribution of bound CK/PCr system (CK) and mitochondria to ATP
supply to the main cellular ATPases (SERC-ATPase and myosinATPase). Numbers represent calculated values of ATP fluxes
from each compartment (see Results for calculations).
Mean⫾SEM of 6 animals. V0 indicates basal respiration without ADP; Vmax,
maximal ADP-stimulated respiration. Acceptor control ratio, Michaelis-Menten
constant (Km) for ADP, and creatine kinase efficacy were calculated as
described in Materials and Methods.
*P⬍0.05, ‡P⬍0.001, compared with control; †P⬍0.01, compared with Km
without creatine within the same group.
whereas oxygen consumption rate was 5.3⫾1.2 nmol O2 ·
min⫺1 · mg dry weight⫺1. Assuming a one-to-one ATP
production per Cr and an ADP/O ratio of ⱕ3, we estimated
that under our conditions ⱖ65⫾4% of the ATP consumed
came from the CK reaction and ⱕ35⫾4% from mitochondria.
However, blocking the CK reaction by eliminating PCr led to
an 80% compensatory activation of mitochondria to 9.7⫾1.5
nmol O2 · min⫺1 · mg dry weight⫺1 (P⬍0.01). Conversely,
inhibiting mitochondria induced a 40% increase in Cr production to 77⫾9 nmol · min⫺1 · mg dry weight⫺1 (P⬍0.05).
Thus, both the mitochondria and CK system are working on
a competitive basis and can compensate for each other.
Mitochondrial Function and Cytoarchitecture in
CKⴚ/ⴚ Mice
Our experiments suggest that in permeabilized fibers of
normal animals, mitochondria can maintain a high ATP/ADP
ratio near the cellular ATPases when CK is not functionally
active. We have further tested this in mice deficient in
cytosolic and mitochondrial CKs (CK⫺/⫺). These mice exhibited a slight cardiac hypertrophy (heart weight per body
weight ratio 6.0⫾0.3 versus 5.0⫾0.1 mg · g⫺1 in control mice,
P⬍0.05). As expected, CK activity was very low in CK⫺/⫺
cardiac tissue (3.7⫾0.7 versus 480⫾45 IU 䡠 g wet weight⫺1 in
control). There was no overexpression of citrate synthase, a
marker of mitochondrial content (146⫾36 and 144⫾18 IU 䡠 g
wet weight⫺1) or adenylate kinase, another ATP-regenerating
enzyme (178⫾19 and 189⫾23 IU 䡠 g wet weight⫺1) in control
versus CK⫺/⫺ mice, respectively. Moreover, basal and maximal respiration rates as well as acceptor control ratio did not
differ between control and CK⫺/⫺ mice (Table). However,
mitochondria from CK⫺/⫺ mice exhibited a higher sensitivity
to external ADP than control. As expected, Cr decreased the
Km for ADP in control mice due to the functional coupling
between translocase and mitochondrial CK, whereas such an
effect was absent in CK⫺/⫺ mice.
Examination of the ultrastructure of cardiac fibers showed
that although in wild-type mice mitochondria are arranged in
longitudinally running columns between strips of contractile
proteins (Figure 5, left), in CK⫺/⫺ mice, mitochondria and
myofilaments show very obvious signs of reorganization.
Kaasik et al
Energetic Crosstalk Between Organelles
157
Figure 5. Ultrastructure of wild-type and
CK⫺/⫺ cardiomyocytes. Left, Ultrastructure of
the wild-type mouse cardiac myocyte. Mitochondria are arranged in longitudinally running columns between parallel strips of myofibrils. Magnification ⫻12 000. Middle,
Ultrastructure of the CK⫺/⫺ mouse cardiac
myocyte. Abundant mitochondria form bulk
regions and fill all the space between myofibrils. Magnification ⫻12 000. Right, Detail of
the interaction of mitochondria with myofibrils in CK⫺/⫺ mice. Magnification ⫻35 000.
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Frequent splitting of myofibrils resulted in formation of
thinner myofilament bundles and their deviation from the
longitudinal direction (Figure 5, middle). Moreover, the
sarcomere structure appeared altered with decreased A-bands.
Abundant mitochondria form bulk regions and fill all the
space between myofibrils. In some places, mitochondria
entering the myofibrils at sites of splitting could be observed
(Figure 5, right).
Our next step was to investigate whether mitochondrial
ATP could maintain calcium uptake and crossbridge cycling
rate in ventricular fibers of CK⫺/⫺ mice. In these experiments
all steps were performed in the absence of PCr to create equal
conditions for control and CK⫺/⫺ fibers. The results demonstrate that mitochondria-supported calcium load was similar
in both groups (Figure 6A). Similarly, the rate constant of
crossbridge cycling in ATP⫹MITO solution did not significantly differ from control mice (107⫾11 versus 132⫾11 s⫺1
in control; Figure 6B), being largely faster than with ATP
alone (38⫾7 s⫺1). However, this last value was significantly
lower in CK⫺/⫺ than in control mice (63⫾4 s⫺1, P⬍0.02).
Thus, in CK-deficient animals, externally added ATP poorly
supported the SR- and myosin-ATPases, whereas mitochondria could support SR calcium load and crossbridge cycling
rate to a similar extent as in control mice.
Discussion
Functional microcompartmentation of intracellular metabolites and substrates due to the spatial distribution of mitochondria and glycolytic complexes has long been recog-
Figure 6. ATP supply to SERC-ATPase or myosin-ATPase in
cardiac fibers of control and CK⫺/⫺ mice. A, Tension-time integrals of tension transients obtained after 5 minutes of loading
with ATP⫹MITO in fibers from wild-type and CK⫺/⫺ mice. B,
Rate constants of crossbridge cycling of fibers working in
ATP⫹MITO obtained from wild-type and CK-deficient animals.
nized.18 –20 In this respect, striated muscle cells represent a
paradigm of spatial organization between energy-producing
and energy-utilizing sites, because muscle cell organization
should match the high and fluctuating energy utilization
needed for contractile work. Mitochondria are the main
providers of energy in oxidative muscle and heart, and
previous studies18 –20 have suggested a direct interaction
between mitochondria and the main ATP-consuming compartments, the SR, and myofibrils. In digitonin-lysed cardiomyocytes, Altschuld et al27 reported that Ca45 uptake in the
presence of ATP was not sensitive to mitochondrial inhibitors, although this possibility was not directly addressed in
their study.
We took advantage of the cell permeabilization technique
to investigate such a possibility.6,21 The present results show
that direct mitochondrial ATP supply and/or ADP withdrawal
can support the kinetic and thermodynamic requirements of
both the myosin ATPase and SERC-ATPase. This mitochondrial ATP supply appears nearly as effective as CK-supplied
ATP and is much more effective than externally supplied
ATP, in sustaining calcium uptake and contractile speed.
Direct Channeling of ATP and ADP
Between Organelles
These results can be explained in view of the highly ordered
and densely packed organization of adult mammalian cardiac
cells. Close proximity of mitochondria, SR, and myofilaments leads to a situation where nucleotide transfer can occur
much more freely between the organelles than to or from the
cytoplasm. The direct channeling of ATP and ADP between
mitochondria- and ATP-utilizing structures such as the SR
and myofilaments establishes a direct crosstalk between
organelles through compartmentation of adenine nucleotides.
Indeed, it has been demonstrated that access of external ADP
to the mitochondrial matrix is greatly impaired in oxidative
muscles (with an apparent Km for ADP largely exceeding its
cytosolic level21), whereas internally produced ADP has
preferential access to mitochondria.28 It needs to be determined whether the efficacy results primarily from facilitation
of ADP withdrawal or ATP supply. Skinned fiber studies,
together with recent mathematical modeling have pointed out
that ADP diffusion is indeed limited in cardiac cells, suggesting that this limited diffusion is the basis of adenine nucleo-
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tide compartmentation.6,21,29 As a result of the low Km of the
ATPases for ATP and the high Ki for ADP, it is probable that
ADP accumulation is the limiting factor for ATPase activity.
Myosin ATPase and SERC-ATPase are two highly regulated
enzymes, whose effective regulation depends on the relief of
substrate limitation and product accumulation. Moreover, SR
calcium ATPase functions in both forward and reverse mode,
and its efficiency is directly dependent on ⌬GATP.30 Indeed,
accumulation of ADP near the active sites slows down
myosin ATPase and crossbridge cycling rate (see VenturaClapier et al6 for review) or impairs SERC-ATPase activity
and calcium uptake.4 Both phosphotransfer kinases and mitochondria, by sharing their products and their substrates with
the ATPases are able to locally control ATP and ADP
concentrations, thus exerting a thermodynamic and kinetic
control over these enzymes.
The corollary of these observations is that there is no
reason to expect a direct correlation between cardiac work
and global ADP concentration as inferred from nuclear
magnetic resonance (NMR) experiments. This is emphasized
by the fact that changes in workload and increases in
metabolic rates can proceed in heart muscle without marked
changes in PCr and adenine nucleotide contents,9,31 supporting the alternative view that compartmentation and highenergy phosphoryl transfer through phosphotransferases regulate metabolic rates. Recently, Joubert et al32 demonstrated
that the discrepancy between forward and reverse CK fluxes
observed in NMR experiments could be explained by a pool
of ATP not participating in the CK reaction. This pool of ATP
representing 20% of total cellular ATP could represent that
part of ATP directly channeled from mitochondria to
ATPases.
Bound CK and Mitochondria in Controlling Local
ATP/ADP Pools
Our results additionally demonstrated that ATP supplied
directly from mitochondria could be nearly as effective as
ATP supplied by CK/PCr. Moreover, inhibiting one of these
mechanisms led to the immediate activation of the other. The
vital importance of the local control of the adenylate pool is
underscored by the existence of multiple coexisting systems.
However, all the systems may not be exactly equivalent,
although they appear to cooperate within the cell. The CK
system appeared more effective than the crosstalk between
organelles because, when both systems were active, two
thirds of the energy production came from bound CKs. In
intact cells, the CK shuttle could be even more important
because the high cytosolic content of CK could take part in
local ATP regeneration. Moreover, as a result of the nearequilibrium nature of the cytosolic CK reaction, it would be
able to spread the energy signal all over the cell, thus ensuring
coordination of the different cellular substructures, whereas
organelle crosstalk would be more spatially restricted. This
sheds light on the observations obtained in rabbit heart that
CK inhibition accelerates the response time of mitochondria
during rapid workload steps.33 Indeed, inhibiting CK would
increase local ADP for mitochondria thus favoring the direct
interorganelle crosstalk, resulting in more rapid mitochondrial stimulation. On the other hand, direct interaction be-
tween mitochondria and SR or myofilaments could be the
physical basis to explain metabolic waves and spots observed
in energy-depleted isolated cardiac cells.34
In fact, the picture may be even more complex because
glycolytic complexes are also associated with myofibrils and
SR and that at least for SR, glycolytic complexes efficiently
participate in energy supply.35,36 Moreover, adenylate kinase,
which is also bound to intracellular organelles and within
mitochondria, can also participate in phosphotransfer in
cardiac cells, particularly when the CK reaction is impaired.37
Thus, highly structured cytoarchitecture involving direct
organelle interaction, compartmentalized phosphotransfer kinases, and bound glycolytic enzymes allows high efficiency
and fine-tuning of energy transduction systems and cardiac
muscle function.
The partial redundancy of local ATP/ADP-controlling
systems in cardiac muscle is well illustrated in CK knockout
mice. It is recognized that CK deletions do not form a serious
obstacle to normal heart function under laboratory conditions.38 Isolated hearts from CK⫺/⫺ mice have comparable
function at baseline and a nearly equal response to a challenging intervention than control mice,8,9 although at the
moderate workload that could be achieved in Langendorffperfused hearts.29 However, because local control of ATP/
ADP close to cellular ATPases has a critical influence on
enzyme activity, alternative ATP/ADP control systems and
compensatory mechanisms should be operating in CK⫺/⫺
mouse heart to overcome diffusion limitation and to preserve
cardiac function at least at moderate levels of activity.
Mitochondrial content, either morphologically, biochemically, or functionally (the present study) determined, was not
altered in CK⫺/⫺ hear, although it was greatly increased in
skeletal muscles.10,38 However, as previously described in
MM-CK null mice,24 the sensitivity of mitochondrial respiration to external ADP was increased in CK⫺/⫺ mice and
could partially compensate for the lack of mitochondrial CK,
by allowing the cytosolic ADP signal to directly reach the
mitochondrial matrix in the absence of channeling through
CK. Moreover, we observed a remarkable reorganization at
the cytoarchitectural level in the hearts of CK⫺/⫺ mice.
Mitochondria reorganized within myofilaments and tended to
decrease diffusion distances, showing that subcellular organization is sensitive to energy deficiency. Cell remodeling,
direct crosstalk between organelles, and increased mitochondrial ADP sensitivity can obviously participate in such
compensatory mechanisms. In addition, we have recently
reported that glycolytic enzymes can also participate in SR
calcium uptake efficiency but not for myofibrillar function.35
Although adenylate kinase was not upregulated in CK⫺/⫺
mice, it could also contribute to intracellular energy fluxes as
a compensatory mechanism when CK is inhibited.37 The
presence of alternative systems taking part in facilitated ADP
diffusion partially explains the mild phenotype observed in
these mice, at least under normal laboratory conditions.
However, it might be anticipated that, although these systems
may appear redundant at rest or during moderate workload in
the heart, it is highly probable that at a higher energy demand,
the different systems able to control local adenylate pools
would have to be additively recruited.
Kaasik et al
Acknowledgments
This work was supported by Institut National de la Santé et de la
Recherche Médicale (INSERM) grants (to M.N., A.K., and E.B.) and
a Federation of European Biochemical Societies (FEBS) grant (to
A.M.). R.V.-C. is supported by “Center National de la Recherche
Scientifique.” We gratefully acknowledge B. Wieringa and F.
Oerlemans for providing the engineered mice. R. Fischmeister is
acknowledged for continuous support.
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Energetic Crosstalk Between Organelles: Architectural Integration of Energy Production
and Utilization
Allen Kaasik, Vladimir Veksler, Ernest Boehm, Marta Novotova, Ave Minajeva and Renée
Ventura-Clapier
Circ Res. 2001;89:153-159; originally published online July 5, 2001;
doi: 10.1161/hh1401.093440
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