Calcium-induced contraction of sarcomeres changes theregulation of mitochondrial respiration in permeabilizedcardiac cellsTiia Anmann1, Margus Eimre2, Andrey V. Kuznetsov3,4, Tatiana Andrienko3, Tuuli Kaambre1,Peeter Sikk1, Evelin Seppet2, Toomas Tiivel1,2, Marko Vendelin3,5, Enn Seppet1 and 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 Km for 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.

FEBS Journal 272 (2005) 3145–3161 ª 2005 FEBS 3145

of 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 mechanism is based on

the length-dependent activation of sarcomere: stretch-

ing of myofibrils by increasing left ventricle filling

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 contraction 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 membrane

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

Contraction and respiration T. Anmann et al.

3146 FEBS Journal 272 (2005) 3145–3161 ª 2005 FEBS

cyanide-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 lM free calcium in the presence of ATP (1 mM) and glu-

tamate (5 mM). (A) Control ‘ghost’ cardiomyocytes preloaded with 5 lM Rhod-2. (B) Cardiomyocytes after the addition of 1 lM free calcium.

(C) Cardiomyocytes after the addition of 3 lM 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 lM free calcium.

Fig. 1. Contraction of permeabilized cardio-

myocyte induced by calcium (1 lM exter-

nally added free Ca2+) in the presence of

ATP (1 mM). 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.

T. Anmann et al. Contraction and respiration

FEBS Journal 272 (2005) 3145–3161 ª 2005 FEBS 3147

calcium 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�). Thedistribution 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 distribution 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 mM, and cytochrome

c was added to a final concentration of 8 lM. (B) Stability of the

mitochondrial inner membrane potential at 3 lM free Ca2+. The

fluorescence intensity of Rhodamine 123 (0.25 lM) in 2 mL of

gently stirred solution B (0.1 lM free Ca2+) containing 5 mM gluta-

mate and 2 mM 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 lM. Arrows show the final concentration of

free calcium in the system.

Contraction and respiration T. Anmann et al.

3148 FEBS Journal 272 (2005) 3145–3161 ª 2005 FEBS

Dis

trib

utio

n fu

nctio

n 1

0.8

0.6

0.4

0.2

00 1 2 3 4 5

Distance, µm

0°45°90°

3

2

1

0

–1

–2

–3–3 –2 –1 0 1 2 3

50%25%

75%

X, µm

Y,µm

C D

A B

Fig. 4. Quantitative analysis of the regular

arrangement of mitochondria in cardiac cells

preloaded with tetramethylrhodamine ethyl

ester (TMRE) (50 nM). 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.

Dis

trib

utio

n fu

nctio

n 1

0.8

0.6

0.4

0.2

00 1 2 3 4 5

Distance, µm

0°45°

90°

3

2

1

0

–1

–2

–3–3 –2 –1 0 1 2 3

50%25%

75%

X, µm

Y, µ

m

C D

A B

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 lM 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.

T. Anmann et al. Contraction and respiration

FEBS Journal 272 (2005) 3145–3161 ª 2005 FEBS 3149

experiments 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 lmCa2+, 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 Km for 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 Km for 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 Km for 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 Km for 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 experiments

in which the skinned cardiac fibers were incubated

with different concentrations of trypsin, at 4 �C, 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

A B

C D

2,5

1,5

0,5

3

2

1

0 0

100

200

300

400

Km

, µM

0

100

200

300

400

Km

, µM

0 1 2 3 4Ca2+, µM

0 1 2 3 4Ca2+, µM

0 1 2 3 4Ca2+, µM

0 1 2 3 4Ca2+, µM

VO

2 m

ax, n

mol

O2*

mg-1

(ww

)*m

in-1

3,5

2,5

1,5

0,5

3

2

1

0VO

2 m

ax, n

mol

O2*

mg-1

(ww

)*m

in-1

SF, ADPSF, ATP

GF, ADPGF, ATP

GF, ADPGF, ATP

SF, ADPSF, 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

Km of respiration. Maximal respiration rates

were reached at 2.0 and 1.5 mM exogen-

ously added ADP and ATP, respectively. The

apparent Km and 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.

Contraction and respiration T. Anmann et al.

3150 FEBS Journal 272 (2005) 3145–3161 ª 2005 FEBS

degradation 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 Km for exogenous ADP is reversible (Fig. 8).

When cardiomyocytes or fibers incubated in the med-

ium with 3 lm free calcium 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, Km ¼ 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 mitochondrial 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 hyper-

contracted state, to treatment with trypsin. The treatment was per-

formed with increasing trypsin concentrations at a low (0.1 lM, d)

and high (3.0 lM, m) calcium concentration in solution B in the

presence of respiratory substrates, glutamate (5 mM) and malate

(2 mM), but not supplemented by BSA, at 4 �C. Higher trypsin con-

centrations are required for a decrease in the Km value for endo-

genous ADP at a high calcium concentration.

T. Anmann et al. Contraction and respiration

FEBS Journal 272 (2005) 3145–3161 ª 2005 FEBS 3151

creatine 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

Km for ATP was decreased from � 280 lm to � 130 lmat 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

A B

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 lM

free calcium. (B) The hypercontraction

shown was induced by increasing the free

calcium concentration to 1.0 lM in the pres-

ence of 2 mM ATP and 10 mM glutamate.

(C) Cardiomyocytes were then transferred

back into solution B that contained 0.1 lM

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 lM free cal-

cium, then fibers were washed twice (7 min

each wash) in solution B containing 0.1 lM

calcium, and the kinetics were measured

again in the presence of 0.1 lM calcium

(return to this calcium concentration is

shown by 0.1*). The average data for three

separate experiments (±SD) are shown.

Contraction and respiration T. Anmann et al.

3152 FEBS Journal 272 (2005) 3145–3161 ª 2005 FEBS

ATPases 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 mM gluta-

mate and 2 mM malate, dashed line) of oxidative phosphorylation in skinned cardiac fibers. Solution B was supplemented with 5 mM phos-

phoenolpyruvate, 0.24 mM NADH, a large excess of pyruvate kinase (PK) (20 IU mL)1) and lactate dehydrogenase (LDH) (20 IUÆmL)1), and

different [Ca2+] (nominally 0, 0.1 and 2 lM) 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 [Ca2+], Km

for ATP without (1.53 ± 0.16 mM) and with (0.79 ± 0.03 mM*) respiratory substrates, respectively. Glut+Mal represents the effect of respir-

atory substrates 10 mM glutamate and 2 mM malate, respectively, and Atr the effect of atractyloside (98 lM). (B) ADP production rates with

0.1 lM free Ca2+, Km without (2.33 ± 0.41 mM) and with (1.15 ± 0.26 mM*) respiratory substrates. (C) ADP production rates with 2 lM free

Ca2+, Km without (1.88 ± 0.32 mM) and with (2.68 ± 0.71 mM) respiratory substrates. To obtain the Km and Vmax 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

00 200 400 600 800

µM

VO

2, n

mol

O2*

mg-1

(WW

)*m

in-1

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 lM free Ca2+

by exogenous ADP (circles) and ATP (squares) in the absence

(white symbols) and presence of 40 mM Na+ (black symbols) is

shown. The change in the value of maximal respiration rate was

not significant. The number of experiments was 4–8.

SF, 0.1 µM Ca2+,+Cr

SF, 2.0 µM Ca2+,+Cr

SF, 0.1 µM Ca2+

SF, 2.0 µM Ca2+

0 200 400 600 800 1000ATP, µM

3

2

1

0

0,5

1,5

2,5

VO

2, n

mol

O2*

mg-

1 (w

w)*

min

-1

Fig. 11. The effect of creatine on the kinetics of respiration of

skinned fibers by exogenous ATP at 0.1 and 2.0 lM free calcium.

At the 0.1 lM calcium concentration, and in the presence of creat-

ine, the Vmax value increased from 1.48 ± 0.15, in the absence of

creatine, to 2.76 ± 0.10, and decreased at 2.0 lM calcium down to

1.64 ± 0.12, but remained still higher than in with 0.1 lM calcium.

The apparent Km decreased from 275 ± 78 lM to 132 ± 25 lM in

the presence of creatine at 0.1 lM calcium and to 108 ± 26 lM at

2.0 lM calcium.

T. Anmann et al. Contraction and respiration

FEBS Journal 272 (2005) 3145–3161 ª 2005 FEBS 3153

clusion 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 activation of contraction

(‘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 interactions 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 Vmax at 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

Km for 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

Contraction and respiration T. Anmann et al.

3154 FEBS Journal 272 (2005) 3145–3161 ª 2005 FEBS

not effectively diffuse into the ICEU. As a result, the

ATP ⁄ADP ratio near ATPases would decrease and

ADP would accumulate in this space, thereby causing

inhibition of ATPases as a result of the decreased free

energy of ATP hydrolysis [56] and inhibition of

MgATPases by MgADP which has a high affinity to

these enzymes (the Ki is close to 200 lm) [57–59]. To

overcome this inhibition, large doses of ATP had to be

added, which explains the high apparent Km for ATP.

In the cells in vivo this inhibition is overcome by effect-

ive supply of phosphocreatine to myofibrillar creatine

kinase via an energy transfer pathway and rapid rep-

hosphorylation of MgADP [25,37,39–41,58].

Notably, when the intracellular MgATPases oper-

ated under steady state conditions, coupled to the

mitochondrial respiration and rephosphorylation of

endogenous ADP into endogenous ATP, the overall

apparent Km for ATP was decreased, as compared to

MgATPases in the absence of oxidative phosphoryla-

tion (compare Fig. 6B and 9B). This effect, first

observed by Kummel in 1988 [57], is explained by an

increased turnover of ADP and ATP inside the ICEUs

owing to the channelling of both nucleotides. Indeed,

if the accumulation of ADP inside the ICEUs is

responsible for the low apparent affinity of ATPase to

ATP, the removal of ADP should decrease the Km

value. That in this process the mitochondria really

consumed ADP became evident from the observation

that the ADP flux from the ICEUs to the PK+phos-

phoenolpyruvate system decreased after switching on

the oxidative phosphorylation (Fig. 9). Similarly, in

skinned fibers, phosphocreatine shifted the apparent

Km for MgATP for relaxation of rigor tension from

300 lm to 10 lm owing to the coupling of myofibrillar

creatine kinase (MM-CK) to myofibrillar ATPase [58].

The observation that the coupling between ATP pro-

duction and consumption persisted in our experiments

despite the presence of the powerful PK+phos-

phoenolpyruvate system that is capable of eliminating

all cytoplasmic ADP (Fig. 9), indicates directly that

the coupling occurs within the ICEUs and that the dif-

fusion of endogenous ADP out of ICEUs is probably

restricted. Fukuda et al. have shown that MgADP

alone is capable of sigmoidally increasing the active

tension in cardiac cells as a result of the formation of

actomyosin–ADP rigor complexes [59]. In the presence

of the exogenous ATP regenerating system and of high

concentrations of ATP, but in the absence of mitoch-

ondrial substrates, the ATP ⁄ADP ratio was diminished

as a result of the local accumulation of a large quan-

tity of endogenous ADP. This occurs because of high

ATP splitting activity and because the exogenous

PK+phosphoenolpyruvate system was unable to

rephosphorylate the ADP produced in the vicinity of

myosin ATPase [37,39]. Clearly, irrespectively of the

cytoplasmic free [Ca2+], the mitochondria, by phos-

phorylating ADP produced by ATPases, were able to

effectively control the [ADP] and maintain a high

[ATP] : [ADP] ratio near the contractile apparatus.

This type of effective crosstalk between mitochondria

and sarcoplasmic reticulum Ca-dependent MgATPase

was shown by Kaasik et al. by measuring the calcium

accumulation in permeabilized fibers [41].

In our experiments, calcium induced contraction and

increased the proportion of ADP that became avail-

able for phosphorylation by the PK+phospho-

enolpyruvate system. At the same time, exogenously

added ADP became more easily available to mitochon-

dria, as indicated by the decreased Km for ADP in the

regulation of respiration. Both of these findings suggest

that maximal sarcomere shortening may lead to disin-

tegration of the structures of ICEU so that the diffu-

sion restriction for adenine nucleotides through its

barriers decreases. This conclusion conforms to our

understanding that the mitochondria and sarcomeres

are structurally tightly linked to each other so that

changes in sarcomere length ultimately lead to corres-

ponding changes in the length of the adjacent mito-

chondria [60]. These structural relationships are strong

enough to modulate the energy transfer and feedback

systems between the ATPases and mitochondria. Very

recently, reversible and remarkable force development

in myofibrils by the changes of mitochondrial func-

tion ⁄morphology has been directly demonstrated in

cardiomyocytes [61]. Our findings clearly show a

reverse (backward) effect of the regulation in mito-

chondrial function by the changes in sarcomere length

during contraction (shortening).

However, the physiological meaning of the effects

shown in this and other work [41,61,62] remains to be

elucidated. Under physiological conditions, with a high

turnover of contractile cycles during high workloads

and as a result of the presence of creatine kinase and

adenylate kinase systems, the channelling of adenine

nucleotides and metabolic signalling occur mostly via

coupled reactions within the energy transfer networks.

This ensures the highest efficiency of work perform-

ance and overcomes the effects of restrictions of ADP

and ATP diffusion within the ICEUs and at the outer

mitochondrial membrane [37,39]. As a result, the rate

of respiration is controlled by the mitochondrial creat-

ine kinase and adenylate kinase reactions and prob-

ably, to some extent, by the direct channelling of

ADP. The structural changes caused by strong sarco-

mere contractions induced by elevated calcium may

influence all these pathways of energy transfer and

T. Anmann et al. Contraction and respiration

FEBS Journal 272 (2005) 3145–3161 ª 2005 FEBS 3155

feedback signalling. Further experiments under more

physiological conditions (for example, with a complete

contraction–relaxation cycle) are necessary to answer

this question.

Experimental procedures

Animals and tissue preparations

Male Wistar rats (300–350 g in weight) were used in all

experiments. The investigation conformed to the Guide for

the Care and Use of Laboratory Animals published by the

National Institutes of Health (NIH Publication no. 85–23,

revised in 1985).

Mitochondria were isolated from rat hearts, as described

previously [63]. Calcium-tolerant myocytes were isolated by

perfusion with a collagenase-containing medium, as des-

cribed previously [64]. Skinned (permeabilized) fibers were

prepared from rat cardiac muscle and from musculus

soleus, according to the method described previously [65].

Solutions

Composition of the solutions used for the preparation of

skinned fibers and for oxygraphy was based on the informa-

tion of the ionic contents in the muscle cell cytoplasm [66].

Solution A contained, in mm: CaK2-EGTA, 1.9;

K2-EGTA, 8.1; MgCl2, 9.5; dithiothreitol, 0.5; potassium

2-(N-morpholino)ethanesulfonate (K-Mes), 50; imidazole,

20; taurine, 20; Na2ATP, 2.5; phosphocreatine, 15; pH 7.0,

adjusted at 25 �C. Solution B (with 0.1 lm free calcium)

contained, in mm: CaK2-EGTA, 1.9; K2-EGTA, 8.1;

MgCl2, 4.0; dithiothreitol, 0.5; K-Mes 100; pH 7.1, adjusted

at 25 �C; imidazole, 20; taurine, 20; K2HPO4, 3. For oxy-

graphy, 5 mm pyruvate (or 5 mm glutamate) and 2 mm

malate were added as respiratory substrates and used as

sodium salts.

Solution B with different free calcium concentrations

(0.2–4.0 lm) was made by adding CaK2-EGTA and

K2-EGTA stock solutions in different ratios that were cal-

culated by winmaxc, according to the scheme described

below (see the section ‘Calculation of free Ca2+ concentra-

tion’ and Appendix I). Other components were the same as

in solution B. The pH of solutions was adjusted to 7.1

before the kinetic experiments. In some experiments with

confocal microscopy, an increasing amount of CaCl2 stock

solution (270 mm) was added into solution B to adjust the

free Ca2+ concentration to 0.1 or 1.0 lm. A decrease in pH

of <0.3 units, according to our direct measurements, as a

result of the addition of Ca2+ into EGTA-containing buf-

fer, was considered to be too small to interfere with the

results of the experiment. This was confirmed by the

absence of any effects of calcium in the ghost cells or fibers

(see below).

Solution KCl contained, in mm: KCl, 125; Hepes, 20;

glutamate, 4; malate, 2; Mg-acetate, 3; KH2PO4, 5; EGTA,

0.4; and dithiothreitol, 0.3; pH 7.0, adjusted at 25 �C and

5 mgÆmL)1 of BSA was added.

All reagents were purchased from Sigma.

Determination of the rate of mitochondrial respir-

ation in isolated mitochondria, permeabilized

cardiomyocytes, and skinned and ‘ghost’ fibers

The steady state rates of oxygen consumption by isolated

mitochondria, permeabilized cardiomyocytes, and skinned

and ‘ghost’ fibers were recorded as a decrease in oxygen

concentration over time by using the two-channel high

resolution respirometer (Oroboros Oxygraph; Paar KG,

Graz, Austria) or by a Yellow Spring Clark oxygen elec-

trode in solution B with different free calcium concentra-

tion, containing respiratory substrates (see section of

Solution) and 5 mgÆmL)1 of fatty acid free BSA. The rate

of mitochondrial respiration was assessed in response to the

addition of ADP or ATP to different final concentrations

at different free calcium concentrations to determine the

apparent kinetic parameters of respiration regulation.

Determinations were carried out at 25 �C, and the solubil-

ity of oxygen was taken as 215 nmolÆml)1 [43]. The method

of calculation of free calcium concentration in solution B is

given below and in the Appendix.

Confocal microscopy

Isolated saponin-permeabilized cardiomyocytes or fibers

were fixed in a Heraeus flexiperm chamber (Heraus, Hanau,

Germany) using a microscope glass cover slide. The fibers

were fixed at both ends between the flexiperm chamber and

a 22 · 50 mm microscope cover slide. Cardiomyocytes were

simply sedimented in these chambers or in LAB-TEKR

chambered microscopic cover-glasses (Nalge-Nunc Interna-

tional, Naperville, IL, USA). Then, 200 lL of respiration

medium was 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 [Ca2+]m, isola-

ted cardiomyocytes or permeabilized myocardial fibers were

preloaded with the fluorescent Ca2+-specific probe, Rhod-2

(Sigma). For this, cells or fibers were incubated for 30 min

at room temperature in the respiration solution B (see Solu-

tions) with freshly added 5 lm 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 543 nm Helium-Neon laser. The laser

output power was set to an average of 1 mW. The Rhod-2

fluorescence and the flavoprotein autofluorescence were

imaged by using a confocal microscope (LSM510NLO;

Zeiss, Jena, Germany) with a ·40 water immersion lens

Contraction and respiration T. Anmann et al.

3156 FEBS Journal 272 (2005) 3145–3161 ª 2005 FEBS

(NA 1.2). The use of such a water immersion lens preven-

ted geometrical aberrations when observing living cells. The

autofluorescence of flavoproteins was excited with the

488 nm line of an Argon laser, and the laser output power

was set to an average power of 8 mW. The fluorescence sig-

nals were collected through a multiline beam splitter with

maximum reflections at 488±10 nm (for rejection of the

488 nm line) and at 543 nm (for rejection of the 543 nm

line). A second 545 nm beam splitter was used to discrimin-

ate the Rhod-2 signal from the flavoprotein signal. Then,

the flavoprotein signal passed through a 505 nm long-pass

filter before being collected through a pinhole (one Airy

disk unit). The Rhod-2 signal was redirected to a 560 nm

long-pass filter before being collected through a pinhole

(one Airy disk unit).

To analyze the intracellular mitochondrial distribution

and mitochondrial inner membrane potential, myocytes or

fibers were incubated for 30 min at room temperature with

50 nm tetramethylrhodamine ethyl ester (TMRE), added to

respiration medium B. Imaging of TMRE fluorescence was

performed as described for the imaging of mitochondrial

calcium.

In isolated mitochondria the membrane potential was

measured spectrofluorometrically, as described previously

[42].

Quantitative analysis of mitochondrial positioning in the

cell was performed using the method developed in our

laboratories and described very recently [33]. In brief, the

confocal images of the cardiac muscle fibers with easily dis-

tinguishable mitochondria preloaded with TMRE (50 nm)

were used. Each image was rotated until the muscle fiber’s

or cell’s long axis was oriented in a vertical direction, as

judged by eye. Next, the centre of the mitochondria were

marked manually, and the distances to the closest neighbor-

ing mitochondria were computed. The statistical analysis

was performed by computing the distribution function of

the distance between the centres of adjacent mitochondria

[33].

Determination of ATPase activity and direct

channelling of ADP to mitochondria

ATPase activity was determined spectrophotometrically by

monitoring the absorbance decrease at 340 nm in a cuvette

containing solution B complemented with 5 mm phos-

phoenolpyruvate, 0.24 mm NADH, a large excess of PK

(20 IU mL)1), LDH and � 3 mg of skinned fibers, in the

absence or presence of 0.1 or 2.0 lm [Ca2+], at 25 �C.The extent of mitochondrial rephosphorylation of the

ADP produced in ATPase reactions was quantified as a

decrease in the ADP flux through the phosphoenolpyru-

vate-PK system after the addition of respiratory substrates

(10 mm glutamate and 2 mm malate). This part of the ADP

flux was considered to be directly channelled from ATPases

to mitochondria. In some experiments the respiratory sub-

strates and atractyloside (98 lm) were subsequently added

after recording the ADP production vs. [ATP] relationships,

to monitor the effect of mitochondria on ADP production

in the same fiber. For the same purpose, in other experi-

ments atractyloside was added to originally respiring fibers.

Protein concentration determination

Protein concentration in mitochondrial preparations was

determined by ELISA using the ELx800 Universal Micro-

plate Reader from Bio-Tek instruments and a bicinchoninic

acid kit (Protein Assay Reagent) from Pierce (Winooski,

Vermont, USA).

Analysis of the experimental results

The values in the figures are expressed as means ± SD.

The apparent Km and maximal respiration rate for exogen-

ous ADP and ATP were estimated by the Michaelis–Men-

ten equation from the nonlinear least squares fit, applying

simple weighting of the experimental data (from measure-

ments of the respiration of skinned and permeabilized ghost

fibers). Statistical comparisons were made by using analysis

of variance (anova) (variance analysis and Fisher test), and

a P-value of <0.05 was taken as the level of significance.

Calculation of the free concentration of Ca2+

Calculations of the composition of EGTA-Ca buffer were

made according to Fabiato & Fabiato [67], first for a total

calcium concentration of 1.878 mm. In our calculations,

dissociation constants of the complexes of Mg2+ with ADP

and ATP were taken from previously published references

[63,68], as described in the Appendix; 10 mm EGTA and

2.26 mm ATP were used as ligands, and 9.5 mm magnesium

and 1.878 or 2.77 mm calcium were used as metals for cal-

culations for solution A. For solution B we replaced

2.26 mm ATP with 1 mm ADP and changed the concentra-

tion of magnesium to 4 mm and added 3 mm phosphate.

The concentration of free calcium, in the case of 1.878 mm

total calcium, was found to be 1.11 · 10)7 m for solution A

and 1.04 · 10)7 m for solution B. In the case of 2.77 mm

total calcium, free calcium was 1.84 · 10)7 m for solution

A and 1.72 · 10)7 m for solution B.

To increase the free calcium concentration in the confo-

cal microscopic experiments, the total EGTA concentration

in solution B was kept constant at 10 mm and the total cal-

cium concentration changed by adding calculated aliquots

of stock solution of 270 mm CaCl2. In some experiments

the binary mixture of different ratios of K2CaEGTA and

K2EGTA was used in buffer preparation and the Ca2+

concentration was not changed after the pH adjustment.

The necessary total calcium concentrations for achieving

the corresponding free calcium concentrations were calcula-

T. Anmann et al. Contraction and respiration

FEBS Journal 272 (2005) 3145–3161 ª 2005 FEBS 3157

ted by using the winmaxc program according to the

scheme described above. Analysis of the calculations

allowed us also to use a simpler empirical formula:

½Ca�total ¼a½Ca�free

bþ ½Ca�free;

where a ¼ 10.0945 ± 0.01406, and b ¼ 0.4574 ± 0.0021;

for these coefficients, [Ca]free is given in lm and [Ca]total in

mm.

Acknowledgements

This work was supported by INSERM, France, and

Estonian Science Foundation grants No. 5515 and

6142, by the Marie Curie Fellowship of the European

Community programme ‘Improving Human Research

Potential and the Socio-economic Knowledge Base’

(M.V., contract No HPMF-CT-2002–01914) and by

grant no. 0182549s03 from the Ministry of Education

and Science of the Estonian Republic. We wish to

acknowledge Y. Usson (Grenoble, France), Mrs

E. Gvozdkova, M. Peitel and Mr H. Vija (Estonia) for

excellent technical assistance.

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Appendix

Corrections introduced into the programme of

calculations of the free [Ca2+] and [Mg2+]

In the solutions used, it is not only the concentration

of [Ca2+]free that is critical but also the concentration

of Mg2+ this ion participates in many reactions in the

form of MgATP and MgADP complexes. Four ligands

used in the solutions ) EGTA, ATP (in solution A),

ADP (in solution B) and phosphate ) have significant

affinity towards Ca2+ and Mg2+ and their influence

has to be taken into account. To calculate the buffer

compositions, temperature, pH and overall ionic

strength also have to be taken into account. Many

important aspects of the preparation of such kind of

buffers were published recently in [69] by the author

of the software we used for our calculations (see

below).

The influence of temperature (van’t Hoff equation,

see below) and pH on the dissociation constants of

the complexes were calculated by the program itself.

Free calcium concentrations were calculated using

winmaxc (Chris Patton, Stanford University; http://

www.stanford.edu/�cpatton/maxc.html), which is

based largely on the algorithm developed by Fabiato

and Fabiato [67].

Many important features have been added to the

program and the concentrations were checked to take

into account not only pH and ionic strength, but also

the complex formation between other ligands and

metals in the solutions. For our calculations, dissoci-

ation constants for ADP and ATP were taken from

Contraction and respiration T. Anmann et al.

3160 FEBS Journal 272 (2005) 3145–3161 ª 2005 FEBS

[68] and [63], as the constants used in the original pro-

gram were found to be invalid.

Afterwards, the constants provided by [63] were also

corrected as the dissociation constant temperature

dependency was not taken into account. Dependence

of the dissociation and deprotonization constants of

MgADP and MgATP from ionic strength is also dif-

ferent than the equation used in program and equa-

tions worked out using modified Debye–Huckel theory

and provided in [68] were used instead. We found

some typographical errors in the table mentioned

above and therefore the relevant information in correc-

ted form is shown in Table 1.

To guarantee the correct values for the conditions

we were using (25�C, pH 7.1, I ¼ 0.170 N or 0.215 N),

we entered all values of the constants and enthalpies

(in kcalÆmol)1) at these conditions and let the program

provide the correction to the values at the conditions it

used internally (20�C, pH 7.0, I ¼ 0.1 N), assuming

that the correction in reverse was symmetrical. Though

we entered corrected values for enthalpies, it was

important to enter values of the constants at the

temperature of the experiment, as later we found that

the dissociation constant temperature dependency

(van’t Hoff equation)

log10 K0 ¼ log10 K þ DH�

1T � 1

T0

� �

2:303R:

where DH is enthalpy, T is temperature, K is the

dissociation constant and R is the universal gas con-

stant.

The formation of ATPH22– and ADPH2– were

assumed to be negligible at pH 7.1 because of their

low pK values and therefore the constants used in the

program (4.039 and 3.924 respectively) were used.

Other constants used in the program based on data

from Fabiato and Fabiato [67] and were left

unchanged.

In a newsletter from July 28, 2002 (http://www.

stanford.edu/�cpatton/mcn072802.htm) the author of

the program highlighted the errors in the dissociation

constants for ADP and ATP and the present ver-

sions (http://www.stanford.edu/�cpatton/files/winmaxc32.

zip) should contain correct constants. The latter state-

ment was verified by us and the compliance between

constants used by us and the corrected constants found

in the program was found to be satisfactory. Neverthe-

less, in our work we continue to use the constants

given by Phillips [68].

The effect of the temperature on the [Ca2+]free and

[Mg2+]free was calculated. In solution A, the [Ca2+]freewas changed from 0.111 lm at 25�C to 0.158 lm at

4�C. In solution B, the change was smaller – from

0.110 lm at 25�C to 0.106 lm at 4�C. The change of

the [Mg2+]free in this temperature range was consid-

ered insignificant.

It was also important to evaluate if the Mg2+ concen-

tration in solution B (4 mm) was enough under the dif-

ferent experimental conditions. We calculated [ADP]freeand [MgADP] concentrations at [ADP]total concentra-

tions from 0.05 )2 mm (eight concentrations) at highest

(9.15 mm [Ca2+]total) and lowest (1.878 mm [Ca2+]total)

Ca2+ concentrations used in the present work and

found that the ratio of the [MgADP] to [ADP]total was

in the range of 0.725–0.830 at both [Ca2+]total concen-

trations. The ratio for the protonated form of MgADP

was at most 0.019 and for [ADP]free from 0.147 to

0.256. The same calculations were performed using

ATP as a ligand and found that at both Ca2+ concen-

trations the ratio of [MgATP] : [ATP]total was in the

region of 0.940 and 0.965, for protonated form, 0.020 in

whole range and from 0.017 to 0.039 for [ATP]free.

Table 1. Thermodynamic data for the ionization and Mg2+ complex formation reactions of ATP and ADP at 25�C over the pH range 5 to 9.

Data in corrected form, based on [68]. I, ionic strength (N).

Reaction pK DH� (kcal mol-1) pK’ Function

ATPH3� $ ATP4� þ Hþ 7.68 ± 0.01 )1.68 ± 0.30 7:68 � 3:56ffiffiI

pþ 4:90I � 0:04

ATP4� þ Mg2þ $ MgATP2� )5.83 ± 0.10 5.10 ± 0.30 �5:83 þ 6:10ffiffiI

p� 8:74I þ ð2:04

ffiffiI

pÞ=ð1 þ 6:02

ffiffiI

pÞ � 0:10

ATPH3� þ Mg2þ $ MgATPH� )3.59 ± 0.12 2.20 ± 1.25 �3:59 þ 4:06ffiffiI

p� 6:36I þ ð2:04

ffiffiI

pÞ=ð1 þ 6:02

ffiffiI

pÞ � 0:12

ADPH2� $ ADP3� þ Hþ 7.20 ± 0.01 )1.37 ± 0.30 7:20 � 2:54ffiffiI

pþ 3:84I � 0:04

ADP3� þ Mg2þ $ MgADP� )4.27 ± 0.10 4.30 ± 0.30 �4:27 þ 4:06ffiffiI

p� 6:36I þ ð2:04

ffiffiI

pÞ=ð1 þ 6:02

ffiffiI

pÞ � 0:10

ADPH2� þ Mg2þ $ MgADPH )2.45 ± 0.20 0.90 ± 1.40 �2:45 þ 2:03ffiffiI

p� 3:34I þ ð2:04

ffiffiI

pÞ=ð1 þ 6:02

ffiffiI

pÞ � 0:20

T. Anmann et al. Contraction and respiration

FEBS Journal 272 (2005) 3145–3161 ª 2005 FEBS 3161