Metabolic consequences of functional complexes of mitochondria, myofibrils and sarcoplasmic reticulum in muscle cells
1 Laboratory of Fundamental and Applied Bioenergetics, INSERM E0221, Joseph
Fourier University, Grenoble, France
2 A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State
University, Moscow, Russia
3 Department of Transplant Surgery, University Hospital Innsbruck,
Innsbruck, Austria
4 Laboratory of Bioenergetics, National Institute of Chemical Physics and
Biophysics, Tallinn, Estonia
5 RFMQ-TIMC Laboratory, UMR 5525 CNRS, Institute Albert Bonniot, Grenoble,
France
6 Institute of Cybernetics, Tallinn, Estonia
* Author for correspondence (e-mail: valdur.saks{at}ujf-grenoble.fr)
Accepted 14 January 2003
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Summary |
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Key words: mitochondria, myofibril, sarcoplasmic reticulum, cardiomyocyte, mitochondrial respiration, muscle, intracellular energetic unit
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Introduction |
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Materials and methods |
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Isolation of mitochondria from cardiac muscle
Mitochondria were isolated from the hearts of Wistar female rats as
described previously (Saks et al.,
1975).
Isolation and culturing of adult cardiac myocytes
Male Wistar rats weighing 300350g were used in all experiments.
Calcium-tolerant myocytes were isolated by perfusion with a
collagenase-containing medium as described previously
(Kay et al., 1997).
Preparation of skinned and `ghost' cardiac muscle fibers
Skinned (permeabilized) fibers were prepared from rat cardiac muscle and m.
soleus according to the method described previously
(Saks et al., 1998a).
Determination of the rate of mitochondrial respiration in isolated
mitochondria, permeabilized cardiomyocytes, skinned and `ghost' fibers
The rates of oxygen uptake were recorded by using the two-channel
high-resolution respirometer (Oroboros Oxygraph, Paar KG, Graz, Austria) or a
Yellow Spring Clark oxygen electrode (Yellow Spring, OH, USA) in solution B,
with different free calcium concentrations, containing respiratory substrates
(see below) and 2mgml-1 bovine serum albumin (BSA). Determinations
were carried out at 25°C, and solubility of oxygen was taken as
215nmolml-1 (Kuznetsov et al.,
1996). The method of calculation of free calcium concentration in
solution B is given below.
Confocal microscopy
Imaging of mitochondria
Isolated saponin-permeabilized cardiomyocytes or fibers were fixed in a
flexiperm chamber (Heraeus, Hanau, Germany) with microscopic glass slide. 200
µl of respiration medium was then immediately added to the chamber. A fully
oxidized state of mitochondrial flavoproteins was achieved by substrate
deprivation and equilibration of the medium with air. To analyze mitochondrial
calcium, isolated cardiomyocytes or permeabilized myocardial fibers were
preloaded with fluorescent Ca2+-specific probe Rhod-2 (Sigma, St
Louis, MO, USA). For this, cells or fibers were incubated for 30min at room
temperature in solution B (see Solutions) with freshly added 5
µmoll-1 Rhod-2. Rhod-2 has a net positive charge, allowing its
accumulation in mitochondria. The fluorescence of Rhod-2 in loaded myocytes or
fibers was excited with a 543nm heliumneon laser. The laser output
power was set to a mean power of 1mW. Rhod-2 fluorescence and flavoproteins
auto-fluorescence were imaged using a confocal microscope (LSM510NLO; Zeiss,
Jena, Germany) with a 40x water immersion lens (NA 1.2). The use of such
a water immersion lens prevented geometrical aberrations when observing living
cells. For co-localization studies of mitochondria and mitochondrial redox
potential analysis, the autofluorescence of flavoproteins was excited with the
488nm line of an argon laser. The laser output power was set to a mean power
of 8mW. The fluorescence signals were collected through a multi-line beam
splitter with maximum reflections at 488±10nm (for rejection of the
488nm line) and at 543nm (for rejection of the 543nm line). A second 545nm
beam splitter was used to discriminate the Rhod-2 signal from the
flavoproteins signal. The flavoproteins signal was then passed through a 505nm
long-pass filter before being collected through a pinhole (one Airy disk
unit). The Rhod-2 signal was redirected to a 560nm long-pass filter before
being collected through a pinhole (one Airy disk unit).
To analyze mitochondrial distribution and mitochondrial inner membrane potential, myocytes or fibers were incubated for 30min at room temperature with 50nmoll-1 tetramethylrhodamine ethyl ester (TMRE) added to solution B. Imaging of TMRE fluorescence was performed as described for imaging of mitochondrial calcium. In control experiments, dissipation of membrane potential was observed after addition of 5 µmoll-1 antimycin A, 4 µmoll-1 carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and 0.5 µmoll-1 rotenone.
Immunofluorescence confocal microscopy
For labelling of a cytoskeletal network in permeabilized fibers, monoclonal
antibodies against ß-tubulin were used. Cells were first washed in
solution B before being fixed with methanol for 5min at 20°C.
Cardiomyocytes or fibers were washed with phosphate-buffered saline (PBS;
Biomedia, Boussens, France) and incubated in 2% (w/v) bovine serum albumin
(BSA) in PBS overnight at 4°C with primary monoclonal antitubulin antibody
(Sigma) at a 1/200 dilution. After washes in PBS, cells were incubated for 3h
in 2% (w/v) PBS/BSA with secondary antibody rhodamine tetramethyl rhodamine
isothiocyanate (TRITC)-conjugated AffiniPure F(ab')2 fragment
donkey anti-mouse IgG at a dilution of 1/50 (Interchim, Montluçon,
France). Cardiomyocytes or fibers were then washed once in PBS and three times
in water. The labelled cells were deposited on glass cover slips and mounted
in a mixture of Mowiol® and glycerol to which
1,4-diazabicyclo-[2,2,2]-octane (Acros Organics, Pittsburgh, PA, USA) was
added to delay photobleaching. Samples were observed by confocal microscopy
(LSM510 NLO; Zeiss) with a plan apo 40x oil immersion objective lens (NA
1.4).
Determination of pyruvate kinase activity
The activity of pyruvate kinase (PK) in stock solutions was assessed at
25°C by a coupled lactate dehydrogenase system. The decrease in NADH
level, in response to the addition of different amounts of PK, was determined
spectrophotometrically in Uvikon 941 plus (Kontron Instruments, Hertfordshire,
UK) in solution B supplemented with 0.3mmoll-1 NADH,
1mmoll-1 phosphoenolpyruvate (PEP), 2mmoll-1 ADP and
45i.u.ml-1 lactate dehydrogenase.
Protein concentration determination
Protein concentration in mitochondrial preparations was determined by
enzyme-linked immunosorbent assay (ELISA) using the ELx800
universal microplate reader (Bio-Tek Instruments, Winooski, VT, USA) and a BSA
kit (protein assay reagent) from Pierce (Rockford, IL, USA).
Solutions
Composition of the solutions used for preparation of skinned fibers and for
oxygraphy was based on the information of the ionic content in the muscle cell
cytoplasm (Godt and Maughan,
1988).
Solution A
1.9mmoll-1 CaK2EGTA, 8.1mmoll-1
K2EGTA, 9.5mmoll-1 MgCl2,
0.5mmoll-1 dithiothreitol (DTT), 50mmoll-1 potassium
2-(N-morpholino)ethanesulfonate (K-Mes), 20mmoll-1
imidazole, 20mmoll-1 taurine, 2.5mmoll-1
Na2ATP, 15mmoll-1 phosphocreatine, adjusted to pH 7.1 at
25°C.
Solution B
1.9mmoll-1 CaK2EGTA, 8.1mmoll-1
K2EGTA, 4.0mmoll-1 MgCl2,
0.5mmoll-1 DTT, 100mmoll-1 K-Mes, adjusted to pH 7.1 at
25°C, 20mmoll-1 imidazole, 20mmoll-1 taurine,
3mmoll-1 K2HPO4. For oxygraphy,
5mmoll-1 pyruvate (or 5mmoll-1 glutamate) and
2mmoll-1 malate were added as respiratory substrates.
Solution KCl
125mmoll-1 KCl, 20mmoll-1 Hepes, 4mmoll-1
glutamate, 2mmoll-1 malate, 3mmoll-1 Mg-acetate,
5mmoll-1 KH2PO4, 0.4mmoll-1 EGTA
and 0.3mmoll-1 DTT, adjusted to pH 7.1 at 25°C, and
2mgml-1 of BSA was added.
Reagents
All reagents were purchased from Sigma (USA) except ATP and ADP, which were
obtained from Boehringer (Mannheim, Germany).
Analysis of the experimental results
The values in tables and figures are expressed as means ± S.D. The
apparent Km for ADP was estimated from a linear regression
of double-reciprocal plots. Statistical comparisons were made using analysis
of variance (ANOVA) and the Fisher test, and P<0.05 was taken as
the level of significance.
Calculations and modeling
Calculation of free Ca2+ concentration
Calculations of the composition of EGTA-Ca buffer were made according to
Fabiato and Fabiato (1979),
first for a total calcium concentration of 1.878mmoll-1. For our
calculations, dissociation constants of complexes of Mg2+ with ADP
and ATP were taken from Phillips et al.
(1966
) and Saks et al.
(1975
). 10mmoll-1
EGTA and 2.26mmoll-1 ATP were used as ligand concentrations, and
9.5mmoll-1 magnesium and 1.878mmoll-1 or
2.77mmoll-1 calcium were used for metals for calculations for
solution A. For solution B, we replaced 2.26mmoll-1 ATP with
1mmoll-1 ADP, decreased the concentration of magnesium to
4mmoll-1 and added 3mmoll-1 phosphate. In the case of
the 1.878mmoll-1 total calcium concentration, the concentration of
free calcium was found to be 1.11x10-7moll-1 for
solution A and 1.04x10-7moll-1 for solution B. In
the case of the 2.77mmoll-1 total calcium concentration, the
concentration of free calcium was found to be
1.84x10-7moll-1 for solution A and
1.72x10-7moll-1 for solution B, confirming our
previous rough predictions.
To increase the free calcium concentration up to 3 µmoll-1,
the total EGTA concentration in solution B was kept constant at
10mmoll-1 and total calcium concentration was increased by adding
calculated aliquots of a stock solution of 270mmoll-1
CaCl2. The necessary total calcium concentrations for achieving
corresponding free calcium concentrations were calculated using the WINMAXC
program (Stanford University, Stanford, CA, USA) according to the scheme
described above. Analysis of the calculations allowed us also to use a simpler
empirical formula:
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At 21°C, pH 7 and an ionic strength of 0.175moll-1, the total calcium concentrations required to obtain the free calcium concentrations of 0.1, 0.4, 1.0, 2.0 and 3.0 µmoll-1 used in the experiments were 1.81, 4.71, 6.93, 8.22 and 8.76mmoll-1, respectively.
Mathematical modeling of heterogeneous ADP diffusion inside
cardiomyocytes
In this study, we used a modified version of our original mathematical
model of compartmentalized energy transfer
(Aliev and Saks, 1997;
Vendelin et al., 2000
). To
study the ADP diffusion only, the concentrations of creatine and
phosphocreatine were assumed to be zero, corresponding to the experimental
conditions without creatine. The reaction rates of all enzymes were reduced
four times in comparison with the data of our earlier publication
(Vendelin et al., 2000
) to
take into account the difference in temperature (25°C was used in the
present study with skinned cardiac fibers instead of 37°C as used
previously). The ATPase activity
(
ATP) in skinned fibers is taken not
to be periodic (due to non-contracting fibers) but to be stationary and
dependent on the concentrations of MgATP and MgADP ([MgATP] and [MgADP],
respectively), according to the equation:
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In this version of the model, we took into account the geometry of skinned
fiber and the boundary conditions imposed in experiments. The permeabilized
cardiac cell was considered as a cylinder 20 µm in diameter (Figs
1,2).
Because of careful separation of fibers before permeabilization, the diameter
of skinned cardiac muscle fibers was close to that of cardiomyocytes
(Fig. 3). This assumption is in
agreement with an observation that the apparent Km for
exogenous ADP is equally very high for isolated cardiomyocytes and skinned
cardiac fibers (Saks et al.,
1991,
1993
;
Kay et al., 1997
; see below).
We assumed that the concentrations of the metabolites within the solution were
uniform due to stirring during the experiments. Using this assumption and
taking into account the small ratio of the diameter to the length of the
fiber, we simulated the diffusion between the fiber and the solution only in
one cross-section. The cross-section was populated with the mitochondria
(diameter 1 µm). Mitochondria were distributed randomly in the
cross-section to fill 25% of the fiber volume. The concentrations of the
metabolites in the solution and the fiber were approximated in the following
way. The relative volumes of fibers and surrounding solution were taken into
account (the ratio was assumed to be 1:1000). Within the fiber, the
concentration of the metabolites in myoplasm and myofibrils was approximated
using the finite elements. The diffusion path of a metabolite was divided into
the following three parts: (1) restricted diffusion from or into (for
endogenous ADP) the solution through the cytoplasmic and myofibrillar space
and into the vicinity of each mitochondria, with an apparent diffusion
coefficient (Dapp); (2) passive diffusion through the
outer mitochondrial membrane; and (3) carrier-mediated exchanges from the
intermembrane space into the mitochondrial matrix. In our simulations, the
concentrations of the metabolites were computed at the nodal points of the
elements. The flux of the metabolites between the mitochondrion and the
myoplasmatic/myofibrillar compartment is determined by permeability of the
outer membrane and by the gradients of metabolite concentration in the
intermembrane space and on the finite element nodes lying on the boundary
between the mitochondrion and myoplasmatic/myofibrillar compartment. The
concentrations of the metabolites (ADP, Pi and ATP) in the mitochondrial
matrix were calculated from their concentrations in the intermembrane space
and by the kinetics of adenine nucleotide and phosphate transport
(Vendelin et al., 2000
).
Respiration rates were calculated as the functions of the metabolite
concentrations in the mitochondrial matrix
(Vendelin et al., 2000
).
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In simulations presented here, we added a description of the pyruvate
kinase reaction rate (PK;
Saks et al., 1994
). Homogenous
distribution of PK was assumed in solution and in myofibrillar and
cytoplasmatic compartments of the permeabilized fibers. In the model, the
apparent diffusion coefficient of a metabolite in the myofibrillar and
cytoplasmatic compartments
(Dapp=DFxD0, where DF is a
diffusion coefficient factor, and D0 is a diffusion
coefficient in the water or in the bulk water phase in cytoplasm) was varied
by giving different values to DF: the degree of inhibition of mitochondrial
respiration by the PKPEP system at different PK activities was computed
for two values of DF: DF=1, representing the Brownian movement in the water,
and DF=10-1.8, found from fitting the experimental data.
The complete model used in these calculations is available online at the following address: http://cens.ioc.ee/~markov/jexpbiol.2003/jexpbiol.model.pdf.
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Results |
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Respiratory characteristics of permeabilized cell systems
The rate of oxidative phosphorylation (mitochondrial ATP production) is
regulated by ADP due to the respiratory control phenomenon
(Chance and Williams, 1956).
The affinity of oxidative phosphorylation for ADP is quantitatively
characterized by an apparent Km for ADP. For isolated
mitochondria in the homogenous suspension, the value of this constant for ADP
in the medium (exogenous ADP) is very low, 1020 µmoll-1,
due to the high permeability of the outer mitochondrial membrane
(Klingenberg, 1970
) and high
affinity of adenine nucleotide translocator for this substrate
(Vignais, 1976
). However, when
mitochondria are studied in the permeabilized cells in situ, the
results are very different (Saks et al.,
1998a
).
Table 1 summarises the
measured affinities of mitochondria for exogenous ADP in different
preparations before (intact permeabilized cells) and after (ghost cells or
fibers) extraction of myosin. In spite of the very small diffusion distances
(mean=10 µm) from the medium into the core of the cells (Figs
1,
2,
3), in all cases the affinities
are very low compared with the very high values of apparent
Km for exogenous ADP (300400
µmoll-1). An important observation shown in
Table 1 is that activation of
the mitochondrial creatine kinase (miCK) reaction decreases the value of
apparent Km for exogenous ADP. This is due to functional
coupling of the miCK reaction to the oxidative phosphorylation via
the adenine nucleotide translocator
(Barbour et al., 1984;
Joubert et al., 2002
; Saks et
al., 1975
,
1994
,
1995
;
Wallimann et al., 1992
;
Wyss and Kaddurah-Daouk,
2000
), which leads to increased local turnover of adenine
nucleotides in mitochondria, effective aerobic phosphocreatine production and
metabolic stability of the heart (Garlid,
2001
; Kay et al.,
2000
; Saks et al.,
1995
). This emphasizes the role of miCK in regulation of
mitochondrial respiration in muscle cells
(Joubert et al., 2002
;
Kay et al., 2000
;
Saks et al., 2001
;
Walsh et al., 2001
).
Further evidence for this kind of local control of respiration by miCK is
provided in Fig. 4, which shows
the oxygraph recordings of mitochondrial respiration in skinned cardiac fibers
when ADP was produced endogenously in the cellular MgATPase reactions in the
presence of 2mmoll-1 MgATP. Endogenous ADP production activates
respiration several times. Subsequent addition of a system competing with
mitochondria for ADP (Gellerich and Saks,
1982), consisting of pyruvate kinase (PK) in high concentrations
and phosphoenolpyruvate (PEP), reduced the respiration rate, but not by more
than 40%. Addition of creatine increased the respiration rate to its maximal
value observed in State 3. This is again due to activation of local production
of ADP by miCK in the mitochondrial intermembrane space, and this locally
produced ADP is totally inaccessible for exogenous PK but is channeled to the
adenine nucleotide translocator and transported into the mitochondrial matrix
(see above).
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Explanation of these experimental data and of the low efficiency of
inhibition of respiration by exogenous PK can be found by using the
mathematical model of ADP diffusion and energy transfer inside the cells (see
Materials and methods). Fig. 5
shows the results of calculations of the respiration rate for two different
situations. First, the diffusion coefficient, D, for ADP and ATP was
taken to be equal to that in the cellular bulk water phase,
D0=145 µm-2s-1
(Aliev and Saks, 1997). In this
case, because of the small diffusion distance and the high rate of diffusion
(high value of D), ATP and ADP are rapidly exchanged between extra-
and intracellular spaces, and ADP produced endogenously in the cellular
MgATPase reactions is very rapidly consumed by PK (solid line in
Fig. 5). The result is that
respiration is effectively suppressed already in the presence of PK at
activities below 5i.u.ml-1 in the medium (solid line in
Fig. 5). These data are in
agreement with our previous conclusions that the Brownian movement of ADP in
water phase across 10 µm is much faster than its metabolic turnover in
heart mitochondria (Saks et al.,
2001
).
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However, the curve for D0 is much lower than the
experimental dependence (Fig.
5, experimental points). To fit the experimental results described
in Figs 4 and
5, we decreased the mean,
apparent diffusion coefficient
(Dapp=DFxD0), inside the cells,
assuming that the high degree of intracellular structural organization (see
Figs 1,
2,
3) may restrict the diffusion
of adenine nucleotides (Saks et al.,
2001). A good fit between the results of the modeling and the
experimental data was observed when the DF approached the value of
10-2 (Fig. 5). This
means that the intracellular diffusion of ADP (and ATP) is likely to be very
heterogeneous and strongly restricted in some areas inside the cells.
Probably, this occurs both at or near the outer mitochondrial membrane and
between the ICEUs (Aliev and Saks,
1997
; Saks et al.,
1994
,
2001
).
It is also known that the high values of apparent Km
for exogenous ADP are significantly decreased from 300350
µmoll-1 to 4070 µmoll-1 by selective
proteolysis (Kuznetsov et al.,
1996; Saks et al.,
2001
). Treatment of permeabilized cardiomyocytes for a short time
with 1 µmoll-1 trypsin also results in rapid disorganisation of
the regular arrangement of mitochondria in cardiomyocytes and a collapse in
the microtubular network (Appaix et al.,
2003
). Thus, evidently under these conditions, the specific
structure of ICEUs is lost and the local intracellular restrictions for ADP
diffusion are eliminated. This may well explain the decrease in apparent
Km for exogenous ADP. In addition, we have shown
previously that, after similar proteolytic treatment, the endogenous ADP
becomes more accessible for the exogenous PK reaction
(Saks et al., 2001
).
The hypothesis of the heterogeneity of intracellular diffusion of ADP related to the structure of ICEUs is consistent with the new surprising findings described below.
The apparent link between sarcomere length and kinetic parameters of
respiration regulation
The results described in Figs
6,
7,
8 show a new interesting
phenomenon an apparent link between sarcomere length and the affinity
of mitochondria for exogenous ADP, measured as an apparent
Km for this substrate in regulation of mitochondrial
respiration in the permeabilized cells in situ. This phenomenon was
observed when the kinetics of regulation of respiration by ADP were studied at
different free calcium concentrations in two systems: permeabilized cardiac
muscle fibers and permeabilized `ghost' fibers after extraction of myosin. The
free calcium concentration was increased from 0.1 µmoll-1 to 3
µmoll-1, which corresponds to the physiological range of
concentrations (Bers, 2001).
Fig. 6 shows that, in the
presence of ATP (or respiratory substrates and ADP), an increase of free
Ca2+ concentration to 3 µmoll-1 results in strong
contraction of sarcomeres and shortening of fiber length in intact
permeabilized cardiac fibers. If the fibers are not fixed, intermyofibrillar
mitochondria seem to fuse as a result of being pressed together; if the fibers
are fixed in flexiperm and contract isometrically, one observes the appearance
of the empty areas and of a rather long distance between mitochondria. In both
cases, the structure of the cell and the structure of ICEUs are deformed.
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Extraction of myosin prevents these Ca2+-induced structural changes (Fig. 7). The removal of a significant proportion of myosin decreased the total MgATPase activity of fibers (measured in the presence of 3mmoll-1 MgATP) from approximately 4.55.0nmol/minmg-1wetmass (initial mass) to 0.91.0nmol/minmg-1wetmass. In ghost fibers, a very regular arrangement of mitochondria with a precise, parallel fixation in the yz plane of cells was observed (direction of fiber orientation perpendicular to the x-axis), giving the impression of a striated pattern for the intracellular distribution of mitochondria. In these ghost fibers, the regular distance between mitochondria, corresponding to sarcomere length, is not changed with alteration of calcium concentration (Fig. 7). Thus, there is no deformation of the internal, modified structure of the ICEUs in the cell.
Fig. 8 shows that,
surprisingly, the apparent Km for exogenous ADP in
regulation of mitochondrial respiration in intact permeabilized fibers
decreases from 350 µmoll-1 to 30 µmoll-1 with
elevation of the free calcium concentration to 3 µmoll-1 and
deformation of the cell structure (Fig.
8A). A decrease in the Vmax of respiration was
also observed (Fig. 8B). None
of these changes are observed in ghost fibers. In spite of removal of myosin
and a 5-fold decrease in the overall MgATPase activity, the apparent
Km for exogenous ADP (349±34 µmoll-1)
is initially (at 0.1 µmoll-1 free calcium concentration) equal
to that of intact permeabilized fibers and always stays above 250
µmoll-1, even when free Ca2+ concentration is
increased to 3 µmoll-1 (Fig.
8A). Vmax does not change either with
alteration of the free Ca2+ concentration
(Fig. 8B; in comparison with
intact permeabilized fibers, Vmax is elevated in ghost
fibers due to extraction of a large proportion of the protein, i.e. myosin).
Stability of all mitochondrial functions in ghost fibers shows that changes in
the free Ca2+ concentration in the range used does not alter the
mitochondria, which might result from a mechanism of the permeability
transition pore (PTP) opening (Lemasters
et al., 1998).
An important conclusion from these data is that their seems to be a direct structural and functional link between sarcomere structure and mitochondrial function, which is in agreement with the concept of ICEUs.
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Discussion |
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It is well known that patterns of metabolic regulation are very different
in different muscle cells (Hochachka and
McClelland, 1997). In the heart, oxygen consumption rate increases
linearly (more than 10 times compared with the resting state) with the
increase in workload without changes in high-energy phosphate, notably
phosphocreatine levels (Williamson et al.,
1976
; Balaban et al.,
1986
). Thus, this is the system of highest efficiency of feedback
regulation. In skeletal muscle, on the other hand, the phosphocreatine level
decreases rapidly, even during short periods of work, and the decline is
faster (correspondingly, post-exercise recovery is slower) in the fast-twitch
glycolytic muscles than in the oxidative slow-twitch skeletal muscle
(Kushmerick et al., 1992
). The
mitochondrial content and its intracellular arrangement are also remarkably
different: fast-twitch glycolytic muscles have a very low number of
mitochondria, which are localized close to the Ttubule systems near the
Z-line of sarcomeres (Ogata and Yamasaki,
1997
), while in slow-twitch oxidative muscles, and especially in
cardiac muscle, mitochondria are localized in the intermyofibrillar space at
the level of the A-band of sarcomeres
(Duchen, 1999
;
Boudina et al., 2002
). These
structural differences are paralleled by differences in functional
characteristics, in particular in the apparent Km for
exogenous ADP (Kuznetsov et al.,
1996
; Burelle and Hochachka,
2002
). Thus, both intracellular arrangement and regulation of
mitochondrial respiration are tissue specific.
This also seems to be true for mitochondrialcytoskeletal
interactions in general. In many types of cells, one sometimes observes rather
vigorous movement of mitochondria due to their interaction with cytoskeletal
elements, such as the microtubular network and actin microfilaments
(Bereiter-Hahn and Voth, 1994;
Leterrier et al., 1994
;
Margineantu et al., 2000
;
Rizzutto et al., 1998; Yaffe,
1999
). In some cases, the molecular bases behind the organellar
movement of microtubules are motor proteins, kinesin and cytoplasmic dynein,
which bind microtubules and transduce chemical energy of ATP into mechanical
work of mitochondrial movement along microtubules
(Yaffe, 1999
). One may think
that, in cardiac cells, the mitochondria have arrived at their proper, fixed
position inside functional complexes with sarcomeres and sarcoplasmic
reticulum (ICEUs) to achieve the most effective regulation of cellular
energetics. Indeed, during cardiac muscle development, intracellular
distribution of mitochondria changes from a chaotic one in the early postnatal
period to a very regular arrangement in the adult muscle
(Tiivel et al., 2000
). Since
interaction with cytoskeleton is mediated by proteins associated with the
outer mitochondrial membrane (Leterrier et
al., 1994
; Smirnova et al.,
1998
; Yaffe,
1999
), it is easily feasible that these proteins also control the
permeability of the voltage-dependent anion (VDAC) channels, of the outer
mitochondrial membrane (Colombini,
1994
) to adenine nucleotides. However, while the collapse of the
microtubular network (Appaix et al.,
2003
) during short proteolysis coincides with disorganisation of
the regular arrangement of mitochondria in cardiac cells and an increase in
the apparent affinity for exogenous ADP in regulation of respiration as a
result of elimination of local restrictions of diffusion, it is not clear if
only (or if at all) the microtubular network participates in distribution of
mitochondria in the cells and which type of cytolinker proteins is associated
with the mitochondrial surface to fix them precisely inside the cells. These
questions are exciting topics for further research.
The data reported in this work and conforming to the existence of ICEUs
(Fig. 9) in the cardiac cells
as a basic pattern of organisation of energy metabolism are in complete
agreement with the recent evidence that mitochondria are morphologically and
functionally heterogenous within the cells
(Collins et al., 2002).
The strong effect of sarcomere contraction on the apparent
Km for exogenous ADP observed in this work
(Fig. 8)shows that structural
connections between mitochondria and sarcomeres inside ICEUs are very
significant. One of the possible explanations of this surprising phenomenon is
that sarcomere contraction results in deformation of the mitochondrial outer
membrane and opening of the VDAC pores to adenine nucleotides. Another
possibility is that significant shortening of sarcomere length changes the
structure of the ICEUs in general and makes the diffusion of exogenous ADP to
mitochondria inside the cells easier. The observation made in this work is in
good agreement with the data by Nozaki et al.
(2001), who observed that in
rat ventricular papillary muscle the mitochondrial length changes according to
changes in sarcomere length during the transition from normoxia to hypoxia.
Nevertheless, it is too early to speculate about the physiological
significance of this observation. Understanding this phenomenon needs new
experimental investigations.
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Acknowledgments |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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Aliev, M. K. and Saks, V. A. (1997). Compartmentalised energy transfer in cardiomyocytes. Use of mathematical modeling for analysis of in vivo regulation of respiration. Biophys. J. 73,428 -445.[Abstract]
Anflous K., Armstrong, D. D. and Craigen, W. J.
(2001). Altered mitochondrial sensitivity for ADP and maintenance
of creatine-stimulated respiration in oxidative striated muscles from
VDAC1-deficient mice. J. Biol. Chem.
276,1954
-1960.
Appaix, F., Kuznetsov, A., Usson, Y., Kay, L., Andrienko, T.,
Olivares, J., Kaambre, T., Sikk, P., Margreiter, R. and Saks, V.
(2003). Possible role of cytoskeleton in intracellular
arrangement and regulation of mitochondria. Exp.
Physiol. 88,175
-190.
Balaban, R. S., Kantor, H. L., Katz, L. A. and Briggs, R. W. (1986). Relation between work and phosphate metabolite in the in vivo paced mammalian heart. Science 232,1121 -1123.[Medline]
Barbour, R. L., Ribaudo, J. and Chan, S. H. P.
(1984). Effect of creatine kinase activity on mitochondrial
ADP/ATP transport. Evidence for functional interaction. J. Biol.
Chem. 259,8246
-8251.
Bereiter-Hahn, J. and Voth, M. (1994). Dynamics of mitochondria in living cells: shape changes, dislocations, fusion and fission of mitochondria. Microsc. Res. Tech. 27,198 -219.[Medline]
Bers, D. (2001). Excitationcontraction Coupling and Cardiac Contraction. Dordrecht: Kluwer Academic Publishers.
Boudina, S., Laclau, M. N., Tariosse, L., Daret, D., Gouverneur, G., Boron-Adele, S., Saks, V. A. and Dos Santos, P. (2002). Alteration of mitochondrial function in a model of chronic ischemia in vivo in rat heart. Am. J. Physiol. 282,H821 -H831.
Braun, U., Paju, K., Eimre, M., Seppet, E., Orlova, E., Kadaja, L., Trumbeckaite, S., Gellerich, F., Zierz, S., Jockusch, H. and Seppet, E. (2001). Lack of dystrophin is associated with altered integration of the mitochondria and ATPases in slow-twitch muscle cells of MDX mice. Biochim. Biophys. Acta 1505,258 -270.[Medline]
Burelle, Y. and Hochachka, P. W. (2002).
Endurance training induces muscle-specific changes in mitochondrial function
in skinned muscle fibers. J. Appl. Physiol.
92,2429
-2438.
Chance, B. and Williams, G. R. (1956). The respiratory chain and oxidative phosphorylation. Adv. Enzymol. 17,65 -134.
Colombini, M. (1994). Anion channels in the mitochondrial outer membrane. Curr. Top. Membr. 42, 73-101.
Collins, T. J., Berridge, M. J., Lipp, P. and Bootman, M. D.
(2002). Mitochondria are morphologically and functionally
heterogenous within cells. EMBO J.
21,1616
-1627.
de Graaf, R. A., Van Kranenburg, A. and Nicolay, K.
(2000). In vivo31P-NMR spectroscopy of ATP
and phosphocreatine in rat skeletal muscle. Biophys.
J. 78,1657
-1664.
Dos Santos, P., Kowaltowski, A. J., Laclau, M., Subramanian, S., Paucek, P., Boudina, S., Thambo, J. B., Tariosse, L. and Garlid, K. (2002). Mechanisms by which opening the mitochondrial ATP- sensitive K(+) channel protects the ischemic heart. Am. J. Physiol. 283,H284 -H295.
Duchen, M. (1999). Contributions of
mitochondria to animal physiology: from homeostatic sensor to calcium
signalling and death. J. Physiol.
516, 1-17.
Dzeja, P. P., Zeleznikar, R. J. and Goldberg, N. D. (1998). Adenylate kinase: kinetic behaviour in intact cells indicates it is integral to multiple cellular processes. Mol. Cell. Biochem. 184,169 -182.[CrossRef][Medline]
Dzeja, P. P., Vitkevicius, K. T., Redfield, M. M., Burnett, J.
C. and Terzik, A. (1999). Adenylate-kinase catalyzed
phosphotransfer in the myocardium: increased contribution in heart failure.
Circ. Res. 84,1137
-1143.
Fabiato, A. and Fabiato, F. (1979). Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J. Physiol. 75,463 -505.
Fontaine, E. M., Keriel, C., Lantuejoul, S., Rigoulet, M., Leverve, X. M. and Saks, V. A. (1995). Cytoplasmic cellular structures control permeability of outer mitochondrial membrane for ADP and oxidative phosphorylation in rat liver cells. Biochem. Biophys. Res. Commun. 213,138 -146.[CrossRef][Medline]
Garlid, K. (2001). Physiology of mitochondria. In Cell Physiology Sourcebook. A Molecular Approach (ed. N. Sperelakis), pp. 139-151. New York, Boston: Academic Press.
Gellerich, F. and Saks, V. A. (1982). Control of heart mitochondrial oxygen consumption by creatine kinase: the importance of enzyme localization. Biochem. Biophys. Res. Commun. 105,1473 -1481.[Medline]
Godt, R. E. and Maughan, D. W. (1988). On the composition of the cytosol of relaxed skeletal muscle of the frog. Am. J. Physiol. 254,C591 -C604.[Medline]
Gudbjarnason, S., Mathes, P. and Raven, K. G. (1970). Functional compartmentation of ATP and creatine phosphate in heart muscle. J. Mol. Cell. Cardiol 1, 325-339.[Medline]
Hochachka, P. W. and McClelland, G. B. (1997).
Cellular metabolic homeostasis during large-scale change in ATP turnover rates
in muscles. J. Exp. Biol.
200,381
-386.
Joubert, F., Mazet, J. L., Mateo, P. and Joubert, J. A.
(2002). 31P NMR detection of subcellular creatine
kinase fluxes in the perfused rat heart: contractility modifies energy
transfer pathways. J. Biol. Chem.
277,18469
-18476.
Kaasik, A., Veksler, V., Boehm, E., Novotova, M., Minajeva, A.
and Ventura-Clapier, R. (2001). Energetic crosstalk between
organelles. Architectural integration of energy production and utilization.
Circ Res. 89,153
-159.
Kay, L., Li, Z., Fontaine, E., Leverve, X., Olivares, J., Tranqui, L., Tiivel, T., Sikk, P., Kaambre, T., Samuel, J. L., Rappaport, L., Paulin, D. and Saks, V. A. (1997). Study of functional significance of mitochondrialcytoskeletal interactions. In vivo regulation of respiration in cardiac and skeletal muscle cells of desmin-deficient transgenic mice. Biochim. Biophys. Acta 1322,41 -59.[Medline]
Kay, L., Nicolay, K., Wieringa, B., Saks, V. and Wallimann,
T. (2000). Direct evidence of the control of mitochondrial
respiration by mitochondrial creatine kinase in muscle cells in situ.
J. Biol. Chem. 275,6937
-6944.
Kinsey, S. T., Locke, B. R., Benke, B. and Moerland, T. S. (1999). Diffusional anisotropy is induced by subcellular barriers in skeletal muscle. NMR Biomed. 12, 1-7.[CrossRef][Medline]
Klingenberg, M. (1970). Mitochondrial metabolite transport. FEBS Lett. 6, 145-154.[CrossRef][Medline]
Kummel, L. (1988). Ca,MgATPase activity of permeabilized rat heart cells and its functional coupling to oxidative phosphorylation in the cells. Cardiovasc. Res. 22,359 -367.[Medline]
Kushmerick, M. J., Meyer, R. A. and Brown, T. B. (1992). Regulation of oxygen consumption in fast and slow-twitch muscle. Am. J. Physiol. 263,C598 -C606.[Medline]
Kuznetsov, A. V., Tiivel, T., Sikk, P., Käämbre, T., Kay, L., Daneshrad, Z., Rossi, A., Kadaja, L., Peet, N., Seppet, E. and Saks, V. A. (1996). Striking difference between slow and fast twitch muscles in the kinetics of regulation of respiration by ADP in the cells in vivo. Eur. J. Biochem. 241,909 -915.[Abstract]
Kuznetsov, A. V., Mayboroda, O., Kunz, D., Winkler, K.,
Schubert, W. and Kunz, W. S. (1998). Functional imaging of
mitochondria in saponin-permeabilized mice muscle fibers. J. Cell
Biol. 140,1091
-1099.
Lemasters, J. J., Nieminen, A. L., Qian, T., Trost, L., Elmore, S. P., Nishimura, Y., Crowe, R. A., Cascio, W. E., Bradham, C. A., Brenner, D. A. and Herman, B. (1998). The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy. Biochim. Biophys. Acta 1366,177 -196.[Medline]
Leterrier, F., Rusakov, D. A., Nelson, B. D. and Linden, M. (1994). Interactions between brain mitochondria and cytoskeleton: evidence for specialized outer membrane domains involved in the association of cytoskeleton-associated proteins to mitochondria in situ and in vitro. Microsc. Res. Tech. 27,233 -261.[Medline]
Liobikas, J., Kopustinskiene, D. M. and Toleikis, A. (2001). What controls the outer mitochondrial membrane permeability for ADP: facts for and against the oncotic pressure. Biochim. Biophys. Acta 1505,220 -225.[Medline]
Margineantu, D., Capaldi, R. A. and Marcus, A. H.
(2000). Dynamics of the mitochondrial reticulum in live cells
using fourier imaging correlation spectroscopy and digital video microscopy.
Biophys. J. 79,1833
-1849.
Menin, L., Panchichkina, M., Keriel, C., Olivares, J., Braun, U., Seppet, E. K. and Saks, V. A. (2001). Macrocompartmentation of total creatine in cardiomyocytes revisited. Mol. Cell. Biochem. 220,149 -159.[CrossRef][Medline]
Milner, D. J., Mavroidis, M., Weisleder, N. and Capetanaki,
Y. (2000). Desmin cytoskeleton linked to muscle mitochondrial
distribution and respiratory function. J. Cell Biol.
150,1283
-1298.
National Institutes of Health (1985). Guide for the Care and Use of Laboratory Animals. NIH Publication No. 85-23. Bethesda, MD, USA: NIH.
Nozaki, T., Kagaya, Y., Ishide, N., Kitada, S., Miura, M., Nawata, J., Ohno, I., Watanabe, J. and Shirato, K. (2001). Interaction between sarcomere and mitochondrial length in normoxic and hypoxic rat ventricular papillary muscles. Cardiovasc. Pathol. 10,125 -132.[CrossRef][Medline]
Ogata, T. and Yamasaki, Y. (1997). Ultra-high resolution scanning electron microoscopy of mitochondria and sarcoplasmic reticulum arrangement in human red, white, and intermediate muscle fibers. Anatom. Rec. 248,214 -223.[CrossRef][Medline]
Opie, L. H. (1998). The Heart. In Physiology, From Cell To Circulation. Third edition. pp. 43-63.Philadelphia: Lippincott-Raven Publishers.
Phillips, R. C., George, P. and Rutman, R. J. (1966). Thermodynamic studies of the formation and ionization of the magnesium(II) complexes of ADP and ATP over the pH range 5 to 9. J. Am. Chem. Soc. 88,2631 -2640.[Medline]
Rizzuto, R., Pinton, P., Carrington, W., Fay, F. S., Fogarty, K.
E., Lifshitz, L. M., Tuft, R. A. and Pozzan, T. (1998). Close
contacts with the endoplasmic reticulum as determinants for mitochondrial
Ca2+ responses. Science
280,1763
-1766.
Saks, V. A., Chernousova, G. B., Gukovsky, D. E., Smirnov, V. N. and Chazov, E. I. (1975). Studies of energy transport in heart cells. Mitochondrial isoenzyme of creatine phosphokinase: kinetic properties and regulatory action of Mg2+ ions. Eur. J. Biochem. 57,273 -290.[Abstract]
Saks, V. A., Belikova, Yu. O. and Kuznetsov, A. V. (1991). In vivo regulation of mitochondrial respiration in cardiomyocytes: Specific restrictions for intracellular diffusion of ADP. Biochim. Biophys. Acta 1074,302 -311.[Medline]
Saks, V. A., Vassilyeva, E. V., Belikova, Yu. O., Kuznetsov, A. V., Lyapina, S. A., Petrova, L. and Perov, N. A. (1993). Retarded diffusion of ADP in cardiomyocytes: Possible role of outer mitochondrial membrane and creatine kinase in cellular regulation of oxidative phosphorylation. Biochim. Biophys. Acta 1144,134 -148.[Medline]
Saks, V. A., Khuchua, Z. A., Vasilyeva E. V., Belikova, Y. O. and Kuznetsov, A. (1994). Metabolic compartmentation and substrate channeling in muscle cells. Role of coupled creatine kinases in in vivo regulation of cellular respiration. A synthesis. Mol. Cell. Biochem. 133/134,155 -192.
Saks, V. A., Kuznetsov, A. V., Khuchua, Z. A., Vasilyeva, E. V., Belikova, Y. O., Kesvatera, T. and Tiivel, T. (1995). Control of cellular respiration in vivo by mitochondrial outer membrane and by creatine kinase. A new speculative hypothesis: possible involvement of mitochondrialcytoskeleton interactions. J. Mol. Cell. Cardiol. 27,625 -645.[Medline]
Saks, V. A., Veksler, V. I., Kuznetsov, A. V., Kay, L., Sikk, P., Tiivel, T., Tranqui, L., Olivares, J., Winkler, K., Wiedemann, F. and Kunz, W. S. (1998a). Permeabilized cell and skinned fiber techniques in studies of mitochondrial function in vivo. Mol. Cell. Biochem. 184,81 -100.[CrossRef][Medline]
Saks, V. A., Dos Santos, P., Gellerich, F. N. and Diolez, P. (1998b). Quantitative studies of enzymesubstrate compartmentation, functional coupling and metabolic channeling in muscle cells. Mol. Cell. Biochem. 184,291 -307.[CrossRef][Medline]
Saks, V. A., Kaambre, T., Sikk, P., Eimre, M., Orlova, E., Paju, K., Piirsoo, A., Appaix, F., Kay, L., Regiz-Zagrosek, V., Fleck, E. and Seppet, E. (2001). Intracellular energetic units in red muscle cells. Biochem. J. 356,643 -657.[CrossRef][Medline]
Seppet, E., Kaambre, T., Sikk, P., Tiivel, T., Vija, H., Kay, L., Appaix, F., Tonkonogi, M., Sahlin, K. and Saks, V. A. (2001). Functional complexes of mitochondria with MgATPases of myofibrils and sarcoplasmic reticulum in muscle cells. Biochim. Biophys. Acta, 1504,379 -395.[Medline]
Smirnova, E., Shurland, D. L., Ryazantsev, S. N. and van der
Blick, A. M. (1998). A human dynamin-related protein controls
the distribution of mitochondria. J. Cell Biol.
143,351
-359.
Tiivel, T., Kuznetsov, A., Kadaya, L., Käämbre, T., Peet, N., Sikk, P., Braun, U., Ventura Clapier, R., Saks, V. and Seppet, E. K. (2000). Developmental changes in regulation of mitochondrial respiration by ADP and creatine in rat heart in situ.Mol. Cell. Biochem. 208,119 -128.[CrossRef][Medline]
Toleikis, A., Liobikas, J., Trumbeckaite, S. and Majiene, D. (2001). Relevance of fatty acid oxidation in regulation of the outer mitochondrial membrane permeability for ADP. FEBS Lett. 509,245 -249.[CrossRef][Medline]
Veksler, V. I., Kuznetsov, A. V., Sharov, V. G., Kapelko, V. I. and Saks, V. A. (1987). Mitochondrial respiratory parameters in cardiac tissue: a novel method of assessment by using saponin-skinned fibers. Biochim. Biophys. Acta 892,191 -196.[Medline]
Veksler, V. I., Kuznetsov, A. V., Anflous, K., Mateo, P., van
Deursen, J., Wieringa, B. and Ventura-Clapier, R. (1995).
Muscle creatine-kinase deficient mice. II Cardiac and skeletal muscles exhibit
tissue-specific adaptation of the mitochondrial function. J. Biol.
Chem. 270,19921
-19929.
Vendelin, M., Kongas, O. and Saks, V. A.
(2000). Regulation of mitochondrial respiration in heart cells
analyzed by reaction-diffusion model of energy transfer. Am. J.
Cell Physiol. 278,C747
-C764.
Vignais, P. (1976). Molecular and physiological aspects of adenine nucleotide transport in mitochondria. Biochim. Biophys. Acta 456,1 -38.[Medline]
Wallimann, T., Wyss, M., Brdiczka, D., Nicolay, K. and Eppenberger, H. (1992). Transport of energy in muscle: the phosphorylcreatine shuttle. Biochem. J. 281, 21-40.[Medline]
Walsh, B., Tonkonogi, M., Soderlund, K., Hultman, E., Saks, V.
and Sahlin, K. (2001). The role of phosphorylcreatine and
creatine in the regulation of mitochondrial respiration in human skeletal
muscle. J. Physiol. 537,971
-978.
Weiss, J. N. and Korge, P. (2001). The
Cytoplasm. No longer a well-mixed bag. Circ. Res.
89,108
-110.
Williamson, J. R., Ford, C., Illingworth, J. and Safer, B. (1976). Coordination of cyclic acid cycle activity with electron transport flux. Circ. Res. 38 (Suppl. I),39 -51.
Wyss, M. and Kaddurah-Daouk, R. (2000).
Creatine and creatinine metabolisme. Physiol. Rev.
80,1107
-1213.
Yaffe, M. P. (1999). The machinery of
mitochondrial inheritance and behaviour. Science
283,1493
-1497.
Yamashita, H., Sata, M., Sugiura, S., Momomura, S., Serizawa, T. and Iizuka, M. (1994). ADP inhibits the sliding velocity of fluorescent actin filaments on cardiac and skeletal myosins. Circ. Res. 74,1027 -1033.[Abstract]