Up to 3 lm free calcium, in the presence of ATP, induced strong contraction of permeabilized cardiomyocytes with intact sarcomeres, accompanied by alterations in mitochondrial arrangemen
Trang 1regulation of mitochondrial respiration in permeabilized cardiac cells
Tiia Anmann1, Margus Eimre2, Andrey V Kuznetsov3,4, Tatiana Andrienko3, Tuuli Kaambre1,
Peeter Sikk1, Evelin Seppet2, Toomas Tiivel1,2, Marko Vendelin3,5, Enn Seppet1and Valdur A Saks1,3
1 Laboratory of Bioenergetics, National Institute of Chemical Physics and Biophysics, Tallinn, Estonia
2 Department of Pathophysiology, University of Tartu, Estonia
3 Laboratory of Fundamental and Applied Bioenergetics, INSERM E0221, Joseph Fourier University, Grenoble, France
4 Department of General and Transplant Surgery, Innsbruck Medical University, Austria
5 Institute of Cybernetics, Tallinn, Estonia
Calcium ions play a central role in the
excitation-contraction coupling in muscle cells [1,2] and
partici-pate in regulating the activities of multiple enzymes
and metabolic systems, including mitochondrial Krebs
cycle dehydrogenases, in many types of cells [2–6] The
presence of sophisticated Ca-transport systems in
mito-chondria allows these organelles to control the calcium
cycle in the cytoplasmic space [7–15] and the lifetime
of the cell, as overload of mitochondria with calcium
results in opening of the mitochondrial permeability
transition pore, which eventually leads to cell death [11–15] It has also been proposed that, owing to the simultaneous activation of the contractile system and mitochondrial enzymes by calcium, the ATP produc-tion is matched to its demand in cells (‘parallel activa-tion’ mechanism) [16–20] However, both experimental and theoretical studies with detailed mathematical modelling of the calcium effects on the mitochondria showed that calcium can induce, by stimulation of the steps of Krebs cycle, only twofold changes in the rate
Keywords
adenine nucleotides; calcium;
cardio-myocytes; intracellular energetic units,
mitochondria
Correspondence
V A Saks, Laboratory of Bioenergetics,
Joseph Fourier University, 2280, Rue de la
Piscine, BP53X – 38041, Grenoble Cedex 9,
France
Fax: +33 4 76514218
Tel: +33 4 76635627
E-mail: Valdur.Saks@ujf-grenoble.fr
(Received 15 March 2005, revised 21 April
2005, accepted 22 April 2005)
doi:10.1111/j.1742-4658.2005.04734.x
The relationships between cardiac cell structure and the regulation of mitochondrial respiration were studied by applying fluorescent confocal microscopy and analysing the kinetics of mitochondrial ADP-stimulated respiration, during calcium-induced contraction in permeabilized cardiomyo-cytes and myocardial fibers, and in their ‘ghost’ preparations (after selective myosin extraction) Up to 3 lm free calcium, in the presence of ATP, induced strong contraction of permeabilized cardiomyocytes with intact sarcomeres, accompanied by alterations in mitochondrial arrangement and
a significant decrease in the apparent Kmfor exogenous ADP and ATP in the kinetics of mitochondrial respiration The Vmax of respiration showed
a moderate (50%) increase, with an optimum at 0.4 lm free calcium and a decrease at higher calcium concentrations At high free-calcium concentra-tions, the direct flux of ADP from ATPases to mitochondria was dimi-nished compared to that at low calcium levels All of these effects were unrelated either to mitochondrial calcium overload or to mitochondrial permeability transition and were not observed in ‘ghost’ preparations after the selective extraction of myosin Our results suggest that the structural changes transmitted from contractile apparatus to mitochondria modify localized restrictions of the diffusion of adenine nucleotides and thus may actively participate in the regulation of mitochondrial function, in addition
to the metabolic signalling via the creatine kinase system
Abbreviations
FCCP, carbonyl cyanide-p-trifluoromethoxy phenylhydrazone; ICEU, intracellular energetic unit; LDH, lactate dehydrogenase; PK, pyruvate kinase; TMRE, tetramethylrhodamine ethyl ester.
Trang 2of mitochondrial oxidative phosphorylation [21–23].
The magnitude of these direct effects of calcium on
mitochondrial respiration is too small to explain the
variations of the respiration rate in the heart cells
in vivo: in the perfused working rat heart, the
respir-ation rate can be enhanced by more than an order
of magnitude (indeed, by a factor of 20) during
work-load changes under conditions of metabolic stability
[24–27] Under physiological conditions in vivo, cardiac
work and respiration are linearly related [24] and both
are governed by the classical Frank–Starling
mechan-ism [28] The Frank–Starling mechanmechan-ism is based on
the length-dependent activation of sarcomere:
stretch-ing of myofibrils by increasstretch-ing left ventricle fillstretch-ing
increases the force of contraction, work performance
and respiration as a result of increased sensitivity of
the thin filaments to calcium [24,28,29] This results in
an increase in the number of active crossbridges,
with-out any significant changes in the cytoplasmic calcium
transients [30–32]
To explain the observed discrepancies (by a factor
of 10) between the direct effects of calcium on the
respiration of mitochondria and changes in the rates
of oxygen consumption in vivo under physiological
conditions of alteration of workloads, in addition to
the effects of calcium, the metabolic channelling of the
endogenous ADP by the organized energy transfer and
signalling networks (the creatine kinase, adenylate
kin-ase and glycolytic systems) has been proposed as a
major signal for regulating mitochondrial respiration
in cardiac cells [25] In myocytes, mitochondria are
arranged in a crystal-like tissue specific pattern [33],
and in oxidative muscle cells, mitochondria form
func-tional complexes with adjacent sarcoplasmic reticulum
and myofibrils, the intracellular energetic units
(ICEUs) [34–41] In these units, the channelling of
ADP by energy transfer networks overcomes local
restrictions of intracellular diffusion of adenine
nucleo-tides [34,39,40] and explains both the linear
relation-ship between workload and respiration and the
phenomenon of metabolic stability [25] Recently, we
have found, in a preliminary study, that structural
changes, caused by the calcium-induced contraction of
sarcomeres in permeabilized cardiac fibers, significantly
modify the kinetic parameters of mitochondrial
respi-ration regulation by exogenous ADP [35] Very similar
data have been reported for rainbow trout muscle cells
[36] In the current study, the structure–function
rela-tionships in cardiac cells have been studied further
with the aim of detailed quantitative analysis of the
structural and functional alterations induced by
chan-ging free calcium concentrations, both in permeabilized
cardiomyocytes and in myocardial fibers The results
show specific sarcomere–mitochondrial structural and functional links as a result of specific cell organization, and are consistent with the theory of a major role of the metabolic signalling mechanisms in the regulation
of mitochondrial respiration
Results
As could be expected, in the control experiments with isolated and permeabilized cardiomyocytes, neither ATP nor calcium (free calcium concentration 1–3 lm) added alone changed the size of the cardiomyocytes, showing an absence of contraction and of any of their nonspecific effects, as well as an absence of intracellu-lar ATP, endogenous substrates and residual ADP in the permeabilized cardiomyocytes (results not shown) However, when ATP (2 mm), or the respiratory sub-strates glutamate or malate and ADP, were present in the medium, the addition of calcium (at a concentra-tion of 1 lm) resulted in a very strong contracconcentra-tion of cardiomyocytes, and the length of cardiomyocytes was decreased by 50% (Fig 1) This strong contraction
of the cells, without subsequent relaxation, is termed
‘hypercontraction’ in this article These changes of cell size were clearly caused by the sarcomere contraction
as, after the extraction of myosin, the shape of the cells remained unaltered following the addition of ATP and calcium, or of mitochondrial substrates, ADP and calcium (Fig 2) In these experiments, the concentra-tion-dependent increase in the fluorescence intensity
of the mitochondrial calcium sensitive probe, Rhod-2 (Fig 2), clearly indicates significant accumulation of calcium in the mitochondrial matrix
It is known that the accumulation of calcium in mitochondria can lead to the opening of the permea-bility transition pore, associated with mitochondrial swelling and rupture of the outer mitochondrial mem-brane [9–15] Therefore, we used several different methods to test pore opening under the conditions of our experiments Figure 3A shows that the addition of ADP activated the respiration of permeabilized fibers
in the presence of 3 lm free calcium, and that the addition of exogenous cytochrome c did not change the respiration rate This means that endogenous cyto-chrome c always stayed in mitochondria and exogen-ous cytochrome c had no access to the intermembrane space, indicating that the outer mitochondrial mem-brane was intact [42] Monitoring of the memmem-brane potential in isolated heart mitochondria by measuring the uptake of Rhodamine 123 (Fig 3B) shows that the single addition of 3 lm free calcium did not change the membrane potential, but the membrane potential collapsed after the addition of an uncoupler, carbonyl
Trang 3cyanide-p-trifluoromethoxy phenylhydrazone (FCCP).
However, when the mitochondria were titrated with
increasing concentrations of calcium for longer than
40 min, the membrane potential started to decrease after
a concentration of 3 lm calcium was reach (Fig 3C)
This is caused by the accumulation of calcium, over
time, from the Ca-EGTA buffer in medium into the
mito-chondrial matrix As the duration of our experiments
was usually less than 40 min, it is unlikely that the
mitochondrial permeability transition pore was open
Analysis of the confocal images of the permeabilized cardiomyocytes and fibers with intact sarcomeres (Figs 4 and 5) shows that hypercontraction completely disorganized the localization of mitochondria within these cells Here we used the quantitative method of image analysis of confocal micrographs, recently devel-oped in our laboratories [33], to analyse the changes in the arrangement of mitochondria observed in skinned muscles fixed at both ends (i.e in isometric condi-tions) In control solution (containing 0.1 lm free
Fig 2 Absence of contraction in ‘ghost’ cardiomyocytes after the addition up to 3 l M free calcium in the presence of ATP (1 m M ) and glu-tamate (5 m M ) (A) Control ‘ghost’ cardiomyocytes preloaded with 5 l M Rhod-2 (B) Cardiomyocytes after the addition of 1 l M free calcium (C) Cardiomyocytes after the addition of 3 l M free calcium A significant increase in the fluorescence intensity of Rhod-2 clearly shows an elevated calcium concentration in the mitochondrial matrix of ‘ghost’ cardiomyocytes, in particular after the addition of 3 l M free calcium.
Fig 1 Contraction of permeabilized
cardio-myocyte induced by calcium (1 l M
exter-nally added free Ca2+) in the presence of
ATP (1 m M ) Mitochondrial localization was
imaged using confocal microscopy from
autofluorescence of mitochondrial
flavopro-teins, as described in the Experimental
procedures Changes in the shape of one
cardiomyocyte (induced by calcium in the
presence of ATP), resulting in its
hyper-contraction, is shown over time.
Trang 4calcium and no ATP) the mitochondria exhibited a
regular distribution (Fig 4A,B), and Fig 4C shows
that the distances between mitochondrial centers taken
from image in Fig 4B were smallest in the direction
transversal to the fiber, whereas the largest distances
were observed in a diagonal direction (angle 45) The
distribution can be presented in a radial plot, where
the average distance between mitochondrial centers is related to the direction between mitochondria In this plot, the distances between the centers are given by the distances from the reference point (coordinates 0,0) plotted in the direction corresponding to each sector (Fig 4D) From inspection of the radial plot (Fig 4D), it is clear that the mitochondrial centers were not distributed randomly, but arranged according
to a strictly regular pattern
The situation was entirely different if fibers with intact sarcomeres were incubated in the presence of ATP and elevated free calcium (3 lm) (Fig 5) If the fiber is fixed by its ends, the elevated calcium leads to
a disorganization of mitochondrial arrangement in the demonstrated case Indeed, this is evident from the dis-tribution function (Fig 5B): the disdis-tribution function
is almost the same, regardless of the direction In the radial plot (Fig 5D), the centres tended to align along
a circle, which is the expected situation if the random distribution of mitochondria takes place The average distances are increased, in this case, if compared to the control (compare Fig 4D and Fig 5D)
Notably, the arrangement of mitochondria in the cells was changed by high calcium concentrations also
in isolated and permeabilized cardiomyocytes (data not shown) As cardiomyocytes are nonfixed cells, hyper-contraction resulted in a decrease of the length of fibers, and the mitochondria were pressed together between hypercontracted myofibrils [35]
In the permeabilized cardiac fibers with intact sar-comeres, in which an increase in the calcium concen-tration induced hypercontraction and disorganization
of the regular intracellular mitochondrial arrangement, calcium induced changes in the kinetics of regulation
of the respiration rate (Fig 6) This concerned mostly the changes in the apparent affinities for the exogenous adenine nucleotides: a very strong decrease in the val-ues of apparent Km, both for exogenous ADP and for ATP, and much smaller changes in the Vmax of respir-ation (Fig 6A,B) Similar changes were observed in
Fig 3 (A) Cytochrome c test of permeabilized cardiac fibers dem-onstrates the intactness of the outer mitochondrial membrane ADP was added to a final concentration of 2 m M , and cytochrome
c was added to a final concentration of 8 l M (B) Stability of the mitochondrial inner membrane potential at 3 l M free Ca 2+ The fluorescence intensity of Rhodamine 123 (0.25 l M ) in 2 mL of gently stirred solution B (0.1 l M free Ca 2+ ) containing 5 m M gluta-mate and 2 m M malate as mitochondrial substrates and 2 mgÆmL)1
of BSA Isolated rat heart mitochondria were added to a final pro-tein concentration of 0.2 mgÆmL)1 (C) Changes of the mitochond-rial inner membrane potential after a gradual increase of free calcium from 0.1 to 3 l M Arrows show the final concentration of free calcium in the system.
Trang 51 0.8 0.6 0.4 0.2 0
Distance, µm
0°
45°
90°
3 2 1 0 –1 –2 –3 –3 –2 –1 0 1 2 3
50%
X, µm
Fig 4 Quantitative analysis of the regular
arrangement of mitochondria in cardiac cells
preloaded with tetramethylrhodamine ethyl
ester (TMRE) (50 n M ) Representative
confocal image of cardiac muscle fiber (A).
Centers of mitochondria were marked with
small black boxes, as shown in (B) On the
basis of this image, distribution function
(subplot C) and radial plot (subplot D) were
found In subplot C, the distribution
func-tions of distance between the centers of
neighboring mitochondria along the fiber
(direction 90), in cross-fiber direction (0),
and in the diagonal direction (45) are shown.
In subplot D, the distance that encloses
25%, 50%, and 75% of neighboring
mitoch-ondrial centers is shown in the radial plot In
this plot, the distance between
mitochond-rial centers is given through the distance
from the reference point (coordinates 0,0)
and the direction is taken equal to the
middle of the corresponding sector Sector
borders are indicated by dashed lines.
1 0.8 0.6 0.4 0.2 0
Distance, µm
0°
45°
90°
3 2 1 0 –1 –2 –3
50%
X, µm
Fig 5 Quantitative analysis of mitochondrial
arrangement after treatment with calcium.
Fibers were preloaded with
tetramethylrhod-amine ethyl ester (TMRE), as described in
the legend to Fig 4, and fixed at both ends
in a flexiperm chamber Mitochondrial
arran-gement was analyzed after the addition of
3 l M free calcium and incubation for 5 min
at room temperature (A) Representative
confocal image of cardiac muscle fiber (B)
Centers of mitochondria were marked with
small black boxes On the basis of this
image, distribution function (subplot C) and
radial plot (subplot D) were found Note that
the distances between mitochondrial
centres are independent from direction This
is clear from inspecting the distribution
function (subplot C) which is similar in all
directions Some increase of the
intermito-chondrial distances is also seen.
Trang 6experiments in which the inhibitor of the
mitochond-rial calcium uniporter, Ruthenium Red, was used to
avoid any accumulation of calcium in the
mitochon-dria and a possible contribution of the PTP opening
into the kinetics of the respiration regulation (results
not shown) The apparent Km for exogenous ADP
decreases by an order of magnitude, from 320 ±
20 lm to 17 ± 3 lm, with an elevation of the free
cal-cium concentration up to 4 lm (Fig 6B) At the same
time, the Vmax values for respiration only showed a
tendency to increase, with a maximum at 0.4 lm
Ca2+, and then to decrease (Fig 6A) Similarly,
the apparent Km for ATP decreased from
286 ± 49 lm to 54 ± 3 lm (Fig 6B) The Vmax value
was always lower than that with ADP and only
minimally changed with alteration in the free calcium
concentration
On the contrary, no changes in the values of the
apparent Kmfor exogenous ADP were found in
perme-abilized ghost cardiac fibers after the extraction of
myosin, when contraction of sarcomere structures was
made impossible (Fig 2) There was only a slight
ten-dency for a decrease of the apparent Km value, which
was not statistically significant Fig 6D Similarly,
there was only a tendency of a decrease in the Vmax
for the respiration with exogenous ADP in ghost fibers
at calcium concentrations higher than 2 lm (Fig 6C)
Remarkably, the apparent Kmfor exogenous ATP in
the regulation of respiration changed in a manner
simi-lar to that for ADP (Fig 6B) Addition of exogenous
ATP activates intracellular ATPases (the kinetics of
this activation is described below) and endogenous ADP production that, in turn, activates respiration It has been observed before [37–39] and is shown in Fig 6B that in the regulation of respiration, the appar-ent Kmfor exogenous ATP is the same as that for exo-genous ADP In both cases it depends, in very similar manner, on the calcium concentration (Fig 6B) In the case of ghost fibers, both are practically independent
of the free calcium concentration (Fig 6D) This com-parison shows very clearly that the observed decrease
in the apparent Kmfor exogenous adenine nucleotides
in the regulation of respiration of permeabilized fibers with intact sarcomeres is related to the changes induced by their contraction These results show also that the direct effects of changes of free calcium on mitochondrial respiration cardiac cells in situ are not significant
An interesting observation is described in Fig 7 It has been described in multiple studies that the appar-ent Km for exogenous ADP in the regulation of mitochondrial respiration in skinned fibers can be effectively decreased by short-term proteolytic treat-ment [43] Figure 7 shows the result of the experitreat-ments
in which the skinned cardiac fibers were incubated with different concentrations of trypsin, at 4C, in solution B containing 0.1 or 3 lm free calcium and ATP, and then the apparent Km values were deter-mined under standard conditions – in the oxygraphic medium containing 0.1 lm free calcium It is clearly seen in Fig 7 that structural changes induced by sarco-mere contraction decreased the rate of the proteolytic
3,5
2,5
1,5
0,5
3
2
1
100 200 300 400
Km
0 100 200 300 400
Km
Ca 2+ , µM
Ca 2+ , µM
Ca 2+ , µM
Ca 2+ , µM
-1 (ww)*min
3,5
2,5
1,5
0,5
3
2
1
0
-1 (ww)*min
SF, ADP
SF, ATP
GF, ADP
SF, ADP
SF, ATP
Fig 6 The effect of free calcium on the regulation of respiration in skinned (A, B) and ‘ghost’ (C, D) fibers by exogenous ADP and ATP The left panel shows the effect of free calcium on the maximal respiration rates and the right panel shows the effect
of free calcium on the values of apparent
K m of respiration Maximal respiration rates were reached at 2.0 and 1.5 m M exogen-ously added ADP and ATP, respectively The apparent Kmand the maximal rates of respiration are shown as means and SD of the data from different experiments and at different concentrations of free calcium Curves in all graphs are illustrative and show the tendency of the effect of free calcium in skinned and ghost fibers The number of independent experiments used to calculate mean values and SD in all groups, was 3–6 See the text for further details.
Trang 7degradation of proteins that participate in the regular
arrangement of mitochondria and contribute in
mecha-nisms resulting in a high apparent Km for exogenous
ADP, possibly being responsible for the restriction of
ADP diffusion within fibers and across the outer
mito-chondrial membrane [34,40]
Relating to the results described in Fig 7 are data
showing that the effect of the free calcium on the
apparent Kmfor exogenous ADP is reversible (Fig 8)
When cardiomyocytes or fibers incubated in the
med-ium with 3 lm free calcmed-ium were placed again into the
solution containing 0.1 lm free calcium, the apparent
Km for exogenous ADP increased again up to 300 lm
(Fig 8D) Figure 8A–C shows that this occurred in
parallel with a significant recovery of the initial shape
of permeabilized cardiomyocytes
Figure 9 shows the results of studies in which the
fluxes of endogenous ADP in the permeabilized cells
were measured continuously by using a
spectrophoto-metric method with the coupled enzyme system
consisting of the pyruvate kinase (PK),
phospho-enolpyruvate and lactate dehydrogenase (LDH) [37,39]
In the absence of mitochondrial substrates, the total
MgATPase activity of permeabilized fibers (flux of
ADP out of fibers; upper curves in Fig 9A–C)
increased with the addition of calcium, and the
reac-tion was characterized by a very high apparent Km(of
between 1 and 2 mm) for ATP Similar parameters of
the total MgATPase reactions, Km¼ 1.60 ± 0.49 mm,
were found by HPLC (results not shown), under
conditions when they were uncoupled from the mitoch-ondrial respiration If, then, the mitochmitoch-ondrial sub-strates glutamate and malate were added to activate respiration, the flux of ADP out of fibers (as measured
by using the coupled enzyme assay) was strongly decreased, and, vice versa, the addition of atractyloside restored the ADP production rate (as detected by using the PK⁄ LDH assay) to the levels seen without the respiratory substrates (Fig 9A) Thus, the differ-ence between the ATPase activities before and after the addition of respiratory substrate gave the flux of endogenous ADP channelled from MgATPases directly into mitochondria (Fig 9A) It can be seen that this channelled flux was highest at resting levels of cytoso-lic free calcium (0.1 lm) (Fig 9B) Hypercontraction
of sarcomeres caused by increasing the calcium con-centration up to 2 lm significantly decreased the flux available to mitochondria; owing to disorganization
of the cellular structure, more ADP produced by ATPases could diffuse to and be captured by the PK+phosphoenolpyruvate system
Among the results reported in this work, the decrease in the Vmax of respiration with an increase in the free calcium concentration (Fig 6A) is of interest, and may be explained by an inhibitory effect of increased calcium concentration in the mitochondrial matrix on the ATP synthase, as reported by Holmu-hamedov et al [44] To check this possibility, we repeated the kinetic experiments at 2 lm free calcium
in the presence of 40 mm Na+, which reversed the inhibitory effect on the ATP synthase by activating the
Ca2+⁄ Na+ exchange mechanism in the experiments
of Holmuhamedov et al [44] The results shown in Fig 10 demonstrate that the contraction-induced decrease of Vmax is not reversed by 40 mm Na+, nei-ther in the case of exogenous ADP nor of exogenous ATP Thus, a decrease in the Vmax of respiration in skinned cardiac fibers is caused by hypercontraction but not by the direct effect of calcium on mitochond-rial respiration This is in concordance also with an insignificant decrease of Vmax in ghost fibers during elevation of the free calcium concentration in the medium (Fig 6C)
The experiments described above were carried out under experimental conditions that are far from normal physiological conditions The first of the nonphysio-logical conditions is the absence of a contraction– relaxation cycle and of muscular work performance, which results in the rapid production of ADP in the myofibrillar actomyosin reaction The second is the absence of creatine required to activate the creatine kin-ase–phosphocreatine energy transfer pathway In many earlier publications, the strong stimulatory effect of
Fig 7 Change in sensitivity of the skinned fibers, when in a
hycontracted state, to treatment with trypsin The treatment was
per-formed with increasing trypsin concentrations at a low (0.1 l M , d)
and high (3.0 l M , m) calcium concentration in solution B in the
presence of respiratory substrates, glutamate (5 m M ) and malate
(2 m M ), but not supplemented by BSA, at 4 C Higher trypsin
con-centrations are required for a decrease in the Kmvalue for
endo-genous ADP at a high calcium concentration.
Trang 8creatine on respiration in skinned fibers, by decreasing
the apparent Km for exogenous ADP, has been
des-cribed [37,39] Figure 11 shows that a very strong
stimu-latory effect of creatine is observed when exogenous
ATP is used In the presence of creatine, the apparent
Kmfor ATP was decreased from 280 lm to 130 lm
at a free calcium concentration of 0.1 lm, and the Vmax
was strongly increased as a result of ADP production in
the local coupled creatine kinase reactions, including
mitochondrial creatine kinase [38–40] Increase of the
free calcium concentration to 2 lm resulted in some
decrease of the Vmax, but its value stayed higher in the
presence of creatine than in the presence of ATP alone
(Fig 11) Under these conditions, the apparent Km for
ADP remained low because of the presence of both
cal-cium and creatine Thus, under physiological conditions mitochondrial respiration is under the control of the creatine kinase system, and this control may be modified
by an increase in the free calcium concentration
Discussion
The results of this study show that in permeabilized cardiac cells, a significant shortening of sarcomeres – hypercontraction – caused by excess free calcium results in a reversible alteration of the regular arrange-ment of mitochondria in the cells, in the changes in the kinetics of regulation of mitochondrial respiration
by exogenous ADP and ATP, and in the direct channelling of endogenous ADP and ATP between
C
D
Fig 8 Reversibility of the calcium-induced contraction of permeabilized cardiomyo-cytes (A) Cells were incubated and mitoch-ondrial flavoproteins were imaged at 0.1 l M free calcium (B) The hypercontraction shown was induced by increasing the free calcium concentration to 1.0 l M in the pres-ence of 2 m M ATP and 10 m M glutamate (C) Cardiomyocytes were then transferred back into solution B that contained 0.1 l M calcium but no ATP and respiratory sub-strates (D) Reversibility of the effects of calcium-induced contraction of cardiomyo-cytes on the kinetics of regulation of mito-chondrial respiration by exogenous ADP The kinetics of respiratory regulation was measured in solution B containing the respiratory substrates and 3 l M free cal-cium, then fibers were washed twice (7 min each wash) in solution B containing 0.1 l M calcium, and the kinetics were measured again in the presence of 0.1 l M calcium (return to this calcium concentration is shown by 0.1*) The average data for three separate experiments (±SD) are shown.
Trang 9ATPases and mitochondria (channelling meaning the
use of ADP or ATP produced without their release
into the medium) Thus, this study demonstrates
strong structure–function relationships between
ATP-producing and ATP-consuming systems [37–40] and
localized restrictions of the intracellular diffusion of
ADP and ATP related to the precise structural
organ-ization of the cell [34,40]
There is abundant information on the effects of
calcium on mitochondria, including its effects on
mito-chondrial respiration, obtained in studies carried out
on isolated mitochondria during the last three decades; these important investigations date back to the work
by Lehninger et al [3,45,46] and later work to those
by Hansford, Denton, McCormack and others [4–10] These studies have recently been extended to in vivo conditions by using confocal imaging and recombinant protein targeting technology [10–16,47–49] The
con-Fig 9 Kinetics of ADP production in dependence of [ATP] in conditions of the absence (continuous line) and presence (with 10 m M gluta-mate and 2 m M malate, dashed line) of oxidative phosphorylation in skinned cardiac fibers Solution B was supplemented with 5 m M phos-phoenolpyruvate, 0.24 m M NADH, a large excess of pyruvate kinase (PK) (20 IU mL)1) and lactate dehydrogenase (LDH) (20 IUÆmL)1), and different [Ca 2+ ] (nominally 0, 0.1 and 2 l M ) at 25 C The curves are produced from data representing the mean values of groups (n ¼ 2–8).
*P < 0.05 compared to the parameter value in the absence of oxidative phosphorylation (A) ADP production rates without free [Ca 2+ ], K m
for ATP without (1.53 ± 0.16 m M ) and with (0.79 ± 0.03 m M *) respiratory substrates, respectively Glut+Mal represents the effect of respir-atory substrates 10 m M glutamate and 2 m M malate, respectively, and Atr the effect of atractyloside (98 l M ) (B) ADP production rates with 0.1 l M free Ca 2+ , Kmwithout (2.33 ± 0.41 m M ) and with (1.15 ± 0.26 m M *) respiratory substrates (C) ADP production rates with 2 l M free
Ca2+, K m without (1.88 ± 0.32 m M ) and with (2.68 ± 0.71 m M ) respiratory substrates To obtain the K m and V max values for each individual measurement the data were fitted to a Michaelis–Menten relationship, and then average values and standard errors were calculated.
1,2
1
0,8
0,6
0,4
0,2
0
200 400 600 800
µM
-1 (WW)*min
1000 1200 1400 1600
ADP, -Na + ADP, +Na + ATP, -Na + ATP, +Na +
Fig 10 The kinetics of regulation of respiration in permeabilized
cardiac fibers by exogenous ADP and ATP and the effect of
Na-acetate Stimulation of mitochondrial respiration at 2 l M free Ca2+
by exogenous ADP (circles) and ATP (squares) in the absence
(white symbols) and presence of 40 m M Na + (black symbols) is
shown The change in the value of maximal respiration rate was
not significant The number of experiments was 4–8.
ATP, µM
3
2
1
0 0,5 1,5 2,5
-1 (ww)*min
Fig 11 The effect of creatine on the kinetics of respiration of skinned fibers by exogenous ATP at 0.1 and 2.0 l M free calcium.
At the 0.1 l M calcium concentration, and in the presence of creat-ine, the V max value increased from 1.48 ± 0.15, in the absence of creatine, to 2.76 ± 0.10, and decreased at 2.0 l M calcium down to 1.64 ± 0.12, but remained still higher than in with 0.1 l M calcium The apparent K m decreased from 275 ± 78 l M to 132 ± 25 l M in the presence of creatine at 0.1 l M calcium and to 108 ± 26 l M at 2.0 l M calcium.
Trang 10clusion from all these studies is that mitochondria
par-ticipate, by rapid uptake and release of calcium, in the
regulation of localized cellular calcium metabolism and
calcium transients in the cytoplasm, and that calcium
controls cell life and death under pathological
condi-tions by controlling the opening of the mitochondrial
permeability transition pore [9–15] The results of
com-prehensive and excellent biochemical studies have
sometimes also led to the conclusion that calcium may
regulate the main function of mitochondria –
respir-ation and ATP production in oxidative
phosphoryla-tion – in parallel with the activaphosphoryla-tion of contracphosphoryla-tion
(‘parallel activation’ mechanism) [6,7,16,19–22] While
in some types of cells with very low energy fluxes the
activation of ATP synthesis by calcium may be
suffi-cient to satisfy the increased energy demand [16–18],
this enthusiasm in extrapolation of important
informa-tion of Ca–mitochondrial interacinforma-tions to support the
hypothesis of ‘parallel’ activation of respiration and
contraction by calcium may not be justified in the case
of cardiac muscle cells Indeed, direct experimental
studies carried out by Territo et al [21,22], on isolated
heart mitochondria, showed that calcium increases the
respiration rate in the state 3 by a factor of 2–2.5, and
the respiration rate is remarkably high already at a
cal-cium concentration of zero This experimental result
was confirmed by Cortassa et al., from calculations
obtained by using an integrated model of cardiac
mito-chondrial energy metabolism and calcium dynamics
[23] Under physiological conditions, the regulation of
contraction and related energy fluxes in the heart is
governed by the classical Frank–Starling mechanism,
according to which the cardiac work and oxygen
con-sumption may be increased by a factor of 15–20 by
increasing the diastolic filling of the left ventricle
[24,28,29] Under these conditions no changes in the
cytoplasmic calcium transients have been found [30–
32] The cellular explanation of the Frank–Starling
mechanism is based on the length-dependent activation
of myofilaments as a result of the increased sensitivity
of the thin filaments to calcium at a greater sarcomere
length [30–32,50–52] This results in changes in the
number of active crossbridges within sarcomeres at a
constant concentration of intracellular free calcium,
and consequently in the alteration of force
develop-ment, MgATP consumption, and MgADP and Pi
pro-duction Apparently, this initiates an effective feedback
metabolic regulation of respiration via energy transfer
networks [25] The results of this study are in favour
of the latter physiological mechanism Indeed, in the
presence of an excess of exogenous ADP when this
substrate is available at a high concentration, in the
case of the ‘parallel activation’ mechanism, the
max-imal respiration rates should be dependent only upon calcium concentration both in permeabilized cardiac fibers with intact sarcomeres and in ghost fibers, and one should expect a strong increase in the respiration rate with an increase in the calcium concentration However, as shown in Fig 6, there is only a slight increase of Vmax (by some 40%), with the optimum free calcium concentration of 0.4 lm, in permeabilized fibers, and a subsequent decrease in Vmaxat higher cal-cium concentrations, and these modest changes in
Vmax are completely eliminated in ghost fibers, from which most of the myosin ATPase is depleted Similar observations have been made previously [35,36] Clearly, calcium ions are unable to stimulate oxidative phosphorylation without involvement of extramito-chondrial ATPases Under conditions of hypercont-raction, in the absence of relaxation and force development, the contraction cycle is probably slowed down and the related actomyosin MgATPase activity decreased, thus decreasing the direct supply of ADP to mitochondria
This conclusion is also consistent with the results of Khuchua et al., who have shown that there is no direct significant activation of mitochondrial respiration by
Ca2+ions in muscle cells in situ [53] but the effects of changes in free calcium concentration rather result from indirect effects of the Ca2+ stimulation of acto-myosin crossbridge cycling that provides ADP to acti-vate respiration [53]
This study shows also that unitary organization of intracellular energy metabolism into ICEUs confers the effective regulative mechanisms of ATPases to car-diac cells This is evident from comparison of the ATPase vs [ATP] relationships in isolated myofibrils and skinned fibers: whereas our analysis revealed the
Kmfor MgATP in the MgATPase reaction to be close
to 1.5–2 mm in saponin-skinned cardiac fibers, the value of Km was more than two orders of magnitude less in isolated myofibrils (10–50 lm) [54,55] in the absence of oxidative phosphorylation In both prepara-tions the PK+phosphoenolpyruvate system was used for measurements of ADP produced by ATPases However, in contrast to isolated myofibrils, where the PK+phosphoenolpyruvate system could effectively eliminate the accumulation of ADP (a product of the ATPase reaction), thereby conferring high ATP-sensi-tivity to myofibrils, the PK+phosphoenolpyruvate system was unable to consume the endogenous ADP produced in the interior space of the ICEUs in skinned fibers, as it has been many times demonstrated [35,37,39] Hence, ADP could accumulate and remain inside the ICEUs owing to restricted diffusion out from that structure For the same reasons, ATP could