Calcium-induced contraction of sarcomeres changes the regulation 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 Seppet1 and Valdur A Saks1,3 Laboratory of Bioenergetics, National Institute of Chemical Physics and Biophysics, Tallinn, Estonia Department of Pathophysiology, University of Tartu, Estonia Laboratory of Fundamental and Applied Bioenergetics, INSERM E0221, Joseph Fourier University, Grenoble, France Department of General and Transplant Surgery, Innsbruck Medical University, Austria Institute of Cybernetics, Tallinn, Estonia Keywords adenine nucleotides; calcium; cardiomyocytes; 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 76514218 Tel: +33 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 cardiomyocytes and myocardial fibers, and in their ‘ghost’ preparations (after selective myosin extraction) Up to 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 concentrations, the direct flux of ADP from ATPases to mitochondria was diminished 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 Calcium ions play a central role in the excitationcontraction coupling in muscle cells [1,2] and participate 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 mitochondria 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 production is matched to its demand in cells (‘parallel activation’ 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 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 Contraction and respiration 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 respiration rate can be enhanced by more than an order of magnitude (indeed, by a factor of 20) during workload 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 mechanism [28] The Frank–Starling mechanism is based on the length-dependent activation of sarcomere: stretching 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, without 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 kinase 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 functional 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 nucleotides [34,39,40] and explains both the linear relationship 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 respiration regulation by exogenous ADP [35] Very similar data have been reported for rainbow trout muscle cells [36] In the current study, the structure–function relationships in cardiac cells have been studied further with the aim of detailed quantitative analysis of the structural and functional alterations induced by changing free calcium concentrations, both in permeabilized cardiomyocytes and in myocardial fibers The results 3146 T Anmann et al 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 intracellular ATP, endogenous substrates and residual ADP in the permeabilized cardiomyocytes (results not shown) However, when ATP (2 mm), or the respiratory substrates glutamate or malate and ADP, were present in the medium, the addition of calcium (at a concentration of 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 concentration-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 permeability transition pore, associated with mitochondrial swelling and rupture of the outer mitochondrial membrane [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 lm free calcium, and that the addition of exogenous cytochrome c did not change the respiration rate This means that endogenous cytochrome c always stayed in mitochondria and exogenous cytochrome c had no access to the intermembrane space, indicating that the outer mitochondrial membrane 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 lm free calcium did not change the membrane potential, but the membrane potential collapsed after the addition of an uncoupler, carbonyl FEBS Journal 272 (2005) 3145–3161 ª 2005 FEBS T Anmann et al Contraction and respiration Fig Contraction of permeabilized cardiomyocyte induced by calcium (1 lM externally added free Ca2+) in the presence of ATP (1 mM) Mitochondrial localization was imaged using confocal microscopy from autofluorescence of mitochondrial flavoproteins, as described in the Experimental procedures Changes in the shape of one cardiomyocyte (induced by calcium in the presence of ATP), resulting in its hypercontraction, is shown over time Fig Absence of contraction in ‘ghost’ cardiomyocytes after the addition up to lM free calcium in the presence of ATP (1 mM) and glutamate (5 mM) (A) Control ‘ghost’ cardiomyocytes preloaded with lM Rhod-2 (B) Cardiomyocytes after the addition of lM free calcium (C) Cardiomyocytes after the addition of 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 lM free calcium 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 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 mitochondrial matrix As the duration of our experiments was usually less than 40 min, it is unlikely that the mitochondrial permeability transition pore was open FEBS Journal 272 (2005) 3145–3161 ª 2005 FEBS Analysis of the confocal images of the permeabilized cardiomyocytes and fibers with intact sarcomeres (Figs 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 developed 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 conditions) In control solution (containing 0.1 lm free 3147 Contraction and respiration T Anmann et al Fig (A) Cytochrome c test of permeabilized cardiac fibers demonstrates the intactness of the outer mitochondrial membrane ADP was added to a final concentration of mM, and cytochrome c was added to a final concentration of lM (B) Stability of the mitochondrial inner membrane potential at lM free Ca2+ The fluorescence intensity of Rhodamine 123 (0.25 lM) in mL of gently stirred solution B (0.1 lM free Ca2+) containing mM glutamate and mM malate as mitochondrial substrates and mgỈmL)1 of BSA Isolated rat heart mitochondria were added to a final protein concentration of 0.2 mgỈmL)1 (C) Changes of the mitochondrial inner membrane potential after a gradual increase of free calcium from 0.1 to lM Arrows show the final concentration of free calcium in the system 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°) The distribution can be presented in a radial plot, where 3148 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 distribution 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, hypercontraction 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 sarcomeres, in which an increase in the calcium concentration 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 values of apparent Km, both for exogenous ADP and for ATP, and much smaller changes in the Vmax of respiration (Fig 6A,B) Similar changes were observed in FEBS Journal 272 (2005) 3145–3161 ª 2005 FEBS T Anmann et al Contraction and respiration B A C D 0.8 0° 45° 90° 0.6 0.4 0.2 0 FEBS Journal 272 (2005) 3145–3161 ª 2005 FEBS Distance, µm –3 –3 –2 –1 X, µm B D C 0.8 0.6 0° 45° 0.4 90° Y, µm Distribution function 25% 50% 75% –1 –2 A Fig Quantitative analysis of mitochondrial arrangement after treatment with calcium Fibers were preloaded with tetramethylrhodamine ethyl ester (TMRE), as described in the legend to Fig 4, and fixed at both ends in a flexiperm chamber Mitochondrial arrangement was analyzed after the addition of lM free calcium and incubation for 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 intermitochondrial distances is also seen Y,µm Distribution function Fig 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 functions 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 mitochondrial centers is shown in the radial plot In this plot, the distance between mitochondrial 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 25% 50% 75% –1 0.2 0 –2 Distance, µm –3 –3 –2 –1 X, µm 3149 Contraction and respiration T Anmann et al B 400 3,5 SF, ADP SF, ATP 2,5 1,5 SF, ADP SF, ATP 200 100 0,5 0 Ca2+, µM C Ca2+, µM D 400 3,5 300 2,5 Km, µM VO2 max, nmolO2*mg-1(ww)*min-1 300 Km, µM VO2 max, nmolO2*mg-1(ww)*min-1 A 1,5 GF, ADP GF, ATP 200 GF, ADP GF, ATP 100 0,5 0 Ca2+, 0 µM experiments in which the inhibitor of the mitochondrial calcium uniporter, Ruthenium Red, was used to avoid any accumulation of calcium in the mitochondria 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 ± lm, with an elevation of the free calcium concentration up to 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 ± 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 permeabilized ghost cardiac fibers after the extraction of myosin, when contraction of sarcomere structures was made impossible (Fig 2) There was only a slight tendency 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 lm (Fig 6C) Remarkably, the apparent Km for exogenous ATP in the regulation of respiration changed in a manner similar to that for ADP (Fig 6B) Addition of exogenous ATP activates intracellular ATPases (the kinetics of 3150 Ca2+, µM Fig 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 exogenously 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 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 apparent Km for exogenous ATP is the same as that for exogenous 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 comparison 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 It has been described in multiple studies that the apparent Km for exogenous ADP in the regulation of mitochondrial respiration in skinned fibers can be effectively decreased by short-term proteolytic treatment [43] Figure shows the result of the experiments in which the skinned cardiac fibers were incubated with different concentrations of trypsin, at °C, in solution B containing 0.1 or lm free calcium and ATP, and then the apparent Km values were determined under standard conditions – in the oxygraphic medium containing 0.1 lm free calcium It is clearly seen in Fig that structural changes induced by sarcomere contraction decreased the rate of the proteolytic FEBS Journal 272 (2005) 3145–3161 ª 2005 FEBS T Anmann et al Fig Change in sensitivity of the skinned fibers, when in a hypercontracted state, to treatment with trypsin The treatment was performed 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 °C Higher trypsin concentrations are required for a decrease in the Km value for endogenous ADP at a high calcium concentration degradation of proteins that participate in the regular arrangement of mitochondria and contribute in mechanisms resulting in a high apparent Km for exogenous ADP, possibly being responsible for the restriction of ADP diffusion within fibers and across the outer mitochondrial membrane [34,40] Relating to the results described in Fig 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 medium with 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 shows the results of studies in which the fluxes of endogenous ADP in the permeabilized cells were measured continuously by using a spectrophotometric method with the coupled enzyme system consisting of the pyruvate kinase (PK), phosphoenolpyruvate 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 reaction was characterized by a very high apparent Km (of between and 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 FEBS Journal 272 (2005) 3145–3161 ª 2005 FEBS Contraction and respiration conditions when they were uncoupled from the mitochondrial respiration If, then, the mitochondrial substrates 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 difference 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 cytosolic free calcium (0.1 lm) (Fig 9B) Hypercontraction of sarcomeres caused by increasing the calcium concentration up to 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 Holmuhamedov et al [44] To check this possibility, we repeated the kinetic experiments at 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+, neither 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 mitochondrial 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 nonphysiological 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 kinase–phosphocreatine energy transfer pathway In many earlier publications, the strong stimulatory effect of 3151 Contraction and respiration A T Anmann et al B C Fig Reversibility of the calcium-induced contraction of permeabilized cardiomyocytes (A) Cells were incubated and mitochondrial 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 presence of 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 substrates (D) Reversibility of the effects of calcium-induced contraction of cardiomyocytes on the kinetics of regulation of mitochondrial respiration by exogenous ADP The kinetics of respiratory regulation was measured in solution B containing the respiratory substrates and lM free calcium, then fibers were washed twice (7 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 D creatine on respiration in skinned fibers, by decreasing the apparent Km for exogenous ADP, has been described [37,39] Figure 11 shows that a very strong stimulatory 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 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 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 cal3152 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 arrangement 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 FEBS Journal 272 (2005) 3145–3161 ª 2005 FEBS T Anmann et al Contraction and respiration Fig Kinetics of ADP production in dependence of [ATP] in conditions of the absence (continuous line) and presence (with 10 mM glutamate and mM malate, dashed line) of oxidative phosphorylation in skinned cardiac fibers Solution B was supplemented with mM phosphoenolpyruvate, 0.24 mM NADH, a large excess of pyruvate kinase (PK) (20 IU mL)1) and lactate dehydrogenase (LDH) (20 ImL)1), and different [Ca2+] (nominally 0, 0.1 and 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 respiratory substrates 10 mM glutamate and 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 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 VO2, nmolO2*mg-1(ww)*min-1 VO2, nmolO2*mg-1(WW)*min-1 0,8 0,6 ADP, -Na+ ADP, +Na+ ATP, -Na+ ATP, +Na+ 0,4 0,2 200 400 600 800 1000 1200 1400 1600 µM Fig 10 The kinetics of regulation of respiration in permeabilized cardiac fibers by exogenous ADP and ATP and the effect of Naacetate Stimulation of mitochondrial respiration at 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 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 ATPproducing and ATP-consuming systems [37–40] and localized restrictions of the intracellular diffusion of ADP and ATP related to the precise structural organization of the cell [34,40] There is abundant information on the effects of calcium on mitochondria, including its effects on mitoFEBS Journal 272 (2005) 31453161 ê 2005 FEBS SF, 2.0 àM Ca2+,+Cr 1,5 SF, 0.1 µM Ca2+ SF, 2.0 µM Ca2+ 0,5 0 SF, 0.1 µM Ca2+,+Cr 2,5 200 400 600 800 1000 ATP, µM 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 creatine, 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 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 con3153 Contraction and respiration clusion from all these studies is that mitochondria participate, 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 conditions by controlling the opening of the mitochondrial permeability transition pore [9–15] The results of comprehensive and excellent biochemical studies have sometimes also led to the conclusion that calcium may regulate the main function of mitochondria – respiration and ATP production in oxidative phosphorylation – 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 sufficient to satisfy the increased energy demand [16–18], this enthusiasm in extrapolation of important information 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 by a factor of 2–2.5, and the respiration rate is remarkably high already at a calcium concentration of zero This experimental result was confirmed by Cortassa et al., from calculations obtained by using an integrated model of cardiac mitochondrial 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 consumption 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 development, MgATP consumption, and MgADP and Pi production 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 max3154 T Anmann et al 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 calcium 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 extramitochondrial ATPases Under conditions of hypercontraction, 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 actomyosin crossbridge cycling that provides ADP to activate respiration [53] This study shows also that unitary organization of intracellular energy metabolism into ICEUs confers the effective regulative mechanisms of ATPases to cardiac 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 preparations 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-sensitivity 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 FEBS Journal 272 (2005) 3145–3161 ª 2005 FEBS T Anmann et al 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 effective supply of phosphocreatine to myofibrillar creatine kinase via an energy transfer pathway and rapid rephosphorylation of MgADP [25,37,39–41,58] Notably, when the intracellular MgATPases operated 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 phosphorylation (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+phosphoenolpyruvate 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 production and consumption persisted in our experiments despite the presence of the powerful PK+phosphoenolpyruvate system that is capable of eliminating all cytoplasmic ADP (Fig 9), indicates directly that the coupling occurs within the ICEUs and that the diffusion 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 mitochondrial substrates, the ATP ⁄ ADP ratio was diminished as a result of the local accumulation of a large quantity of endogenous ADP This occurs because of high ATP splitting activity and because the exogenous PK+phosphoenolpyruvate system was unable to FEBS Journal 272 (2005) 3145–3161 ª 2005 FEBS Contraction and respiration rephosphorylate the ADP produced in the vicinity of myosin ATPase [37,39] Clearly, irrespectively of the cytoplasmic free [Ca2+], the mitochondria, by phosphorylating 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 available for phosphorylation by the PK+phosphoenolpyruvate system At the same time, exogenously added ADP became more easily available to mitochondria, 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 disintegration of the structures of ICEU so that the diffusion 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 corresponding changes in the length of the adjacent mitochondria [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 function ⁄ morphology has been directly demonstrated in cardiomyocytes [61] Our findings clearly show a reverse (backward) effect of the regulation in mitochondrial 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 performance 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 creatine kinase and adenylate kinase reactions and probably, to some extent, by the direct channelling of ADP The structural changes caused by strong sarcomere contractions induced by elevated calcium may influence all these pathways of energy transfer and 3155 Contraction and respiration feedback signalling Further experiments under more physiological conditions (for example, with a complete contraction–relaxation cycle) are necessary to answer this question T Anmann et al 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 mgỈmL)1 of BSA was added All reagents were purchased from Sigma 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 described 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 information 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, For oxygraphy, mm pyruvate (or mm glutamate) and 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 calculated by winmaxc, according to the scheme described below (see the section ‘Calculation of free Ca2+ concentration’ 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 buffer, 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) 3156 Determination of the rate of mitochondrial respiration 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 electrode in solution B with different free calcium concentration, containing respiratory substrates (see section of Solution) and mgỈmL)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 solubility of oxygen was taken as 215 nmolỈml)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 International, 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, isolated 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 at room temperature in the respiration solution B (see Solutions) with freshly added 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 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 FEBS Journal 272 (2005) 3145–3161 ª 2005 FEBS T Anmann et al (NA 1.2) The use of such a water immersion lens prevented 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 mW The fluorescence signals 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 discriminate 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 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 distinguishable 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 neighboring 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 mm phosphoenolpyruvate, 0.24 mm NADH, a large excess of PK (20 IU mL)1), LDH and % 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 phosphoenolpyruvate-PK system after the addition of respiratory substrates (10 mm glutamate and mm malate) This part of the ADP flux was considered to be directly channelled from ATPases to mitochondria In some experiments the respiratory sub- FEBS Journal 272 (2005) 3145–3161 ª 2005 FEBS Contraction and respiration 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 experiments atractyloside was added to originally respiring fibers Protein concentration determination Protein concentration in mitochondrial preparations was determined by ELISA using the ELx800 Universal Microplate 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 exogenous ADP and ATP were estimated by the Michaelis–Menten equation from the nonlinear least squares fit, applying simple weighting of the experimental data (from measurements 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 calculations for solution A For solution B we replaced 2.26 mm ATP with mm ADP and changed the concentration of magnesium to mm and added 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 confocal microscopic experiments, the total EGTA concentration in solution B was kept constant at 10 mm and the total calcium 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- 3157 Contraction and respiration T Anmann et al 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: ẵCatotal ẳ aẵCafree ; b ỵ ẵCafree 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 References Bers D (2001) Excitation-Contraction Coupling and Cardiac Contraction Kluwer Academic Publishers, Dordrecht Berridge MJ, Bootman MD & Roderick HL (2003) 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complexes of ADP and ATP over the pH range 5–9 J Am Chem Soc 88, 2631–2640 69 Patton C, Thompson S & Epel D (2004) Some precautions in using chelators to buffer metals in biological solutions Cell Calcium 35, 427–431 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, dissociation constants for ADP and ATP were taken from FEBS Journal 272 (2005) 3145–3161 ª 2005 FEBS T Anmann et al Contraction and respiration Table Thermodynamic data for the ionization and Mg2+ complex formation reactions of ATP and ADP at 25°C over the pH range to Data in corrected form, based on [68] I, ionic strength (N) Reaction 3À DH° (kcal mol-1) pK ỵ ATPH $ ATP ỵ H ATP4 ỵ Mg2ỵ $ MgATP2 ATPH3 ỵ Mg2ỵ $ MgATPH ADPH2 $ ADP3 ỵ Hỵ ADP3 ỵ Mg2ỵ $ MgADP ADPH2 ỵ Mg2ỵ $ MgADPH 7.68 )5.83 )3.59 7.20 )4.27 )2.45 ± ± ± ± ± 0.01 0.10 0.12 0.01 0.10 0.20 )1.68 5.10 2.20 )1.37 4.30 0.90 [68] and [63], as the constants used in the original program 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 different than the equation used in program and equations worked out using modied DebyeHuckel theory ă and provided in [68] were used instead We found some typographical errors in the table mentioned above and therefore the relevant information in corrected form is shown in Table 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 kcalỈmol)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) À1 Á  T À T0 : log10 K ¼ log10 K þ DH 2:303R where DH is enthalpy, T is temperature, K is the dissociation constant and R is the universal gas constant 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 FEBS Journal 272 (2005) 3145–3161 ª 2005 FEBS ± ± ± ± ± ± 0.30 0.30 1.25 0.30 0.30 1.40 pK’ Function pffi 7:68 À 3:56 I ỵ 4:90I ặ 0:04 p p p 5:83 þ 6:10 I À 8:74I þ ð2:04 IÞ=ð1 þ 6:02 Iị ặ 0:10 p p p 3:59 ỵ 4:06 I 6:36I ỵ 2:04 Iị=1 ỵ 6:02 Iị ặ 0:12 p 7:20 2:54 I ỵ 3:84I ặ 0:04 p p p 4:27 ỵ 4:06 I 6:36I ỵ 2:04 Iị=1 ỵ 6:02 Iị ặ 0:10 p p p 2:45 þ 2:03 I À 3:34I þ ð2:04 IÞ=ð1 þ 6:02 Iị ặ 0:20 from Fabiato and Fabiato [67] and were left unchanged In a newsletter from July 28, 2002 (http://www stanford.edu/$cpatton/mcn072802.htm) the author of the program highlighted the errors in the dissociation constants for ADP and ATP and the present versions (http://www.stanford.edu/$cpatton/files/winmaxc32 zip) should contain correct constants The latter statement 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 Nevertheless, 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+]free was 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 considered insignificant It was also important to evaluate if the Mg2+ concentration in solution B (4 mm) was enough under the different experimental conditions We calculated [ADP]free and [MgADP] concentrations at [ADP]total concentrations 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 concentrations 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+ concentrations 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 3161 ... results in a reversible alteration of the regular arrangement of mitochondria in the cells, in the changes in the kinetics of regulation of mitochondrial respiration by exogenous ADP and ATP, and in. .. 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... recovery of the initial shape of permeabilized cardiomyocytes Figure shows the results of studies in which the fluxes of endogenous ADP in the permeabilized cells were measured continuously by using