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* U-446 INSERM, Laboratoire de Cardiologie Cellulaire et Moléculaire, Faculté de Pharmacie, Université Paris-Sud, Châtenay-Malabry, France; and
Department of Pharmacology, Centre of Molecular and Clinical Medicine, University of Tartu, Estonia
1Correspondence: U-446 INSERM, Faculté de Pharmacie, Université Paris-Sud, 5 rue J-B Clément, 92296 Châtenay-Malabry, France. E-mail: Vladimir.VEKSLER{at}cep.u-psud.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: myocardial contractility • mitochondria • myofibrils • potassium
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Structural contacts between the sarcoplasmic reticulum and mitochondria are involved in the control of Ca2+ homeostasis (2
3
4)
and regulation of ATP production (5)
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The present study was designed to reveal functional interactions between mitochondria and myofibrils. We used skinned fibers, which provide an excellent means to investigate the interactions between different compartments (6
7
8)
. Permeabilization of specific membranes with detergents enables a study of organelle function while maintaining cellular architecture and controlling the intracellular medium. As in living cells, mitochondria in these preparations use respiratory substrates for oxidation and can efficiently generate ATP despite the loss of sarcolemmal integrity. By use of this experimental approach, we recently demonstrated direct energetic cross-talk between mitochondria and the ATPases of the sarcoplasmic reticulum and myofibrils (7
, 8)
. These findings suggest that the pool of adenine nucleotides circulating between mitochondria and these ATPases has little communication with the "bulk" cytosolic milieu. This cross-talk evidently benefits from the close proximity of mitochondria and their "energetic partners": the sarcoplasmic reticulum and myofibrils. Here, we show that the functional state of mitochondria can strongly modulate the force developed by myofibrils via a novel mechanism that does not directly involve regulatory pathways identified previously involving Ca2+ or ATP homeostasis. Our data suggest that this regulation may occur via a direct interaction of mitochondria with myofibrils, thus providing evidence in favor of a new principle of mechanical signaling between intracellular compartments.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Individual rat ventricular myocytes were obtained as described (9)
and maintained at 37°C.
Mechanical experiments
Fibers were tied at both ends with a natural silk thread and mounted to a force transducer (AE 801, Aker’s Microelectronics, Horton, Norway). Fibers were immersed in 2.5 mL chambers arranged around a disc, which sat in a 22°C water bath with a magnetic stirrer. At the beginning of each experiment, the fiber was stretched to 120% of its slack length, which corresponded to sarcomere lengths of 2.1–2.2 µm according to laser diffraction estimation (10 mW He-Ne laser, Spectra Physics Inc., Mountain View, CA, USA). Each experiment began by equilibrating the fibers in basic solution in the virtual absence of Ca2+ (pCa 9)
Confocal microscopy
Mitochondrial membrane potential (
m) was estimated in plated permeabilized ventricular myocytes loaded with the m-sensitive fluorescent dye JC-1. Plated myocytes were permeabilized at room temperature for 3 min using basic solution that contained saponin (50 µg/mL), then myocytes were loaded in darkness for 15 min with 1 µmol/L JC-1 in the presence of different drugs that modulated mitochondrial function. Dishes were mounted on the stage of a laser scanning confocal microscope (Carl Zeiss LSM-510) and images were collected with a Plan Apochromat 63x oil immersion objective lens (NA 1.4). Cells were illuminated with the 488 nm line of an Argon laser and emission was monitored using band-pass and long-path emission filters (BP 505-550 and LP 585 nm).
To visualize mitochondrial swelling, cells were loaded for 30 min at room temperature with 200 nmol/L MitoTracker Green. Images were acquired before and after treatment with various mitochondrial modulators for 10–15 min using the 488 nm line of an Argon laser for excitation and an LP505 nm filter for detecting emission. The file names for acquired images were encoded to avoid bias; all images were later subjected to morphometric analysis using the Computer Assisted Stereology Toolbox software (Olympus, Ballerup, Denmark). Fluorescent and nonfluorescent areas in each image were determined by superimposing a grid of points on the images of MitoTracker stained cells, then measuring the percentage of points that overlay fluorescent sites within cells (excluding nuclei), according to standard stereological methods. Seven to 10 cells for each group were analyzed.
Finally, flavin adenine dinucleotide was imaged using the 488 nm line of the Argon laser and a BP 505–550 nm emission filter. These experiments were performed to confirm that changes in MitoTracker fluorescence were due to changes in mitochondrial shape rather than to dye leakage driven by the altered membrane potential.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A). Surprisingly, inhibition of mitochondrial cytochrome c oxidase and ATP synthase by Na azide led to a marked and sustained augmentation of isometric force. This effect was reversible, since activation of the mitochondria by azide withdrawal caused a decline in force to pre-azide levels. The mean increase in force due to mitochondrial de-energization was 39 ± 10% (n=5). This modulation of force development also occurred when fibers were initially activated under conditions of mitochondrial de-energization and then azide was withdrawn (Fig. 1B
). Azide did not affect Ca2+-activated force production in Triton-skinned fibers, in which all membrane structures were destroyed (data not shown).
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It is well known that concentrations of calcium and adenine nucleotides are important regulators of myofibrillar function, so we determined whether the above effect was related to local Ca2+ uptake by energized mitochondria in the vicinity of the myofibrils or to local changes in ATP concentration. Addition of 10 µmol/L ruthenium red, an inhibitor of the mitochondrial Ca2+ uniporter, did not abolish the mechanical response to mitochondrial de-energization. (To avoid possible deleterious effects of prolonged exposure of mitochondria to high Ca2+ concentrations, subsequent experiments were carried out in the presence of 10 µmol/L ruthenium red.) Similarly, 20 µmol/L thapsigargin, an inhibitor of sarcoplasmic reticulum Ca2+ uptake, or 30 µmol/L CGP37157, an inhibitor of the mitochondrial Na+/Ca2+ exchanger, were without effect (data not shown). We then tested the effect of mitochondrial energization on rigor tension, that is, the force developed in the virtual absence of Ca2+ and adenine nucleotides. Figure 1C
shows that in the presence of azide, removal of ATP and PCr induced rigor tension, which was decreased upon activation of mitochondria by azide withdrawal in a manner similar to that observed in the presence of Ca2+ and ATP. The mean value for this decrease was 35 ± 4% (n=5). In these experiments, 25 µmol/L bongkrekic acid, a selective inhibitor of adenine nucleotide transport across the inner mitochondrial membrane, was added to eliminate the possibility that, upon activation, mitochondria might phosphorylate residual ADP compartmentalized in their vicinity. Thus, it can be concluded that the mechanical response of contractile apparatus to mitochondrial activation was related neither to Ca2+ uptake by mitochondria nor to ATP synthesis or hydrolysis; i.e., it was not related to changes in the ATP, ADP, or phosphate near myofibrils.
Mechanical response and m
Since mitochondrial de-energization induces both inhibition of ATP production and dissipation of the transmembrane proton gradient, the next series of experiments sought to determine whether the fiber mechanical response was associated with m changes. Electrochemical potential was measured in situ in saponin-permeabilized rat ventricular cells by confocal microscopy using the potential-sensitive dye JC-1. Much evidence indicates that the chief properties of mitochondria are similar in permeabilized fibers and permeabilized cells. Confocal images show the same regular arrangement of mitochondria between myofibrillar stripes in both preparations and distances between the mitochondrial rows are similar (10)
. Permeabilized fibers and cells demonstrate similar patterns of flavoprotein autofluorescence and calcium-sensitive probe fluorescence in the mitochondria (11)
. Furthermore, functional parameters of mitochondria in permeabilized fibers and cells (sensitivity to ADP, response to creatine) are quite similar (12)
.
As shown in Fig. 2
A, B, m was dissipated by addition of azide. After mitochondrial energization by azide withdrawal, which always led to a decrease in isometric force production, different modulators of mitochondrial function were tested for their effects on contraction and m (Fig. 2C-I
). Bongkrekic acid was used to inhibit adenine nucleotide transport, thereby increasing m, by blocking the process of ADP phosphorylation (Fig. 2C
). As shown, bongkrekic acid potentiated the mechanical effect of mitochondrial energization. Next we tested the effects of dissipating m using a mitochondrial uncoupling agent, the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), together with oligomycin (in order to block mitochondrial ATPase, which is able to maintain the membrane potential at the expense of ATP in the mitochondrial matrix). As shown in Fig. 2D
, FCCP + oligomycin quickly restored a high contractile force, similar to azide. These results further demonstrate that the reduction in contractile force due to mitochondrial activation was not related to ATP production (and/or ADP consumption) by mitochondria, and instead suggest a role for the mitochondrial electrochemical potential in this effect. It is noteworthy that the mechanical response was not related to the opening of the permeability transition pore because cyclosporin A (20 µmol/L), a classical inhibitor of this high-conductance inner membrane channel, had no effect (data not shown).
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How can m of energized mitochondria influence the mechanical properties of the contractile apparatus without involving cytosolic ATP or Ca2+ changes? A likely hypothesis is that this effect is mediated by mitochondrial transmembrane ion fluxes controlled by the electrical potential. As Ca2+ obviously was not involved, we determined whether K+, the most abundant cytoplasmic cation, played a role.
Mitochondrial potassium modulators and mechanical response
First, we tested whether the mechanical response to mitochondrial de-energization occurred in the absence of potassium ions. Replacement of K+ by another cation, tetraethyl ammonium (TEA+), considerably diminished azide-induced force depression (Fig. 3
), However, this substitution had a direct effect on the contractile apparatus: in Triton-skinned fibers, which lack mitochondria, TEA+ markedly reduced force development (Fig. 3
, inset). This observation complicates the interpretation of the effects of TEA+ on mitochondria-myofibril interactions.
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The next series of experiments was designed to test the effects of specific potassium flux modulators. Figure 2E
shows that the K+-selective ionophore valinomycin, which dissipated the electrical potential by causing a massive K+ influx, removed the mechanical effect of mitochondrial activation. By contrast, nigericin, an electroneutral H+/K+ exchanger that reduced the chemical potential by dissipating the proton gradient at the expense of K+ efflux, did not augment the depressed force (Fig. 2F
). This latter result excludes a significant role for protons exported from the mitochondrial matrix in inhibiting contractile force upon mitochondrial energization. Further experiments confirmed the participation of K+ transmembrane fluxes in the mitochondria-dependent force response. Pinacidil (200 µmol/L), which opens mitochondrial ATP-dependent K+ channels, increased contractile force when mitochondria were energized (Fig. 2G
). Diazoxide (200 µM), another opener of these channels, induced the same effect (data not shown). NS-1619, an opener of the recently described mitochondrial Ca2+-activated K+ channels (13)
, also augmented contractile force (Fig. 2H
). Finally, quinine, a blocker of the mitochondrial K+/H+ exchanger that ensures efflux of K+ from the matrix after mitochondrial energization (14)
, strongly increased isometric force production (Fig. 2I
). Measurement of the membrane potential by JC-1 fluorescence showed that none of these modulators of K+ flux eliminated m (Fig. 2G-I
), demonstrating that loss of this potential per se was not necessary for the increase in contractile force.
Mitochondrial geometry and mechanical response
The next aim was to determine how transmembrane K+ fluxes could influence the force developed by cardiomyocytes. It is generally agreed that the main physiological role of transmembrane K+ fluxes is the regulation of mitochondrial volume (15
, 16)
; therefore, we examined whether, under our experimental conditions, the mitochondrial modulators changed the morphology of mitochondria. Figure 4
and Figure 5
show that all of the drugs that favored K+ accumulation in the matrix and increased contractile force (valinomycin, NS-1619, quinine) also induced a clear enlargement of the fluorescent area of mitochondria stained with MitoTracker Green. The boundaries between mitochondria sometimes became indistinguishable; fluorescent areas were shifted closer to lipid droplets, delineating the outline of these droplets. The stripes of space mainly occupied by myofibrils between the mitochondrial rows became more narrow. The most prominent mitochondrial swelling was found during treatment with quinine; 2 mM propranolol (another blocker of the K+/H+ exchanger) had the same effect (data not shown). Other effectors (FCCP+oligomycin, azide), which increased the force of contraction, led to mitochondrial swelling. As expected, the azide-induced mitochondrial swelling was reversible (Fig. 4G
). The mitochondrial adenine nucleotide translocator inhibitor bongkrekic acid did not induce mitochondrial swelling (Fig. 4H
), nor did it increase contractile force (Fig. 2C
). Altogether, these results show that, under various experimental conditions, an increased contractile force is associated with swollen mitochondria.
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Since mitochondrial rows are packed next to the myofibrils, we hypothesized that mitochondrial swelling imposes physical constraints on myofibrils such that it affects the contractile response. In some respects this would resemble the Frank-Starling relationship, in which cell stretch increases isometric force production by imposing mechanical constraints on the myofibrillar compartment. To test this hypothesis, we investigated the contractile response of mitochondrial activation under conditions known to influence the length dependence of force generation.
Mitochondrial energization and properties of the contractile machinery
Cardiac muscle stretch increases maximal contractile force and myofibrillar Ca2+ sensitivity (17)
; therefore, we compared the myofibrillar Ca2+ sensitivity of fibers with de-energized (azide-treated) and energized (without azide) mitochondria. To avoid any possible influence of ATP-generating activity by the mitochondria, the adenine nucleotide translocase was inhibited with bongkrekic acid. Figure 6
shows that mitochondrial de-energization led to a significant increase in the Ca2+ sensitivity of force development.
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The stretch dependence of contractile force development was shown to be reduced considerably by ADP, which may act directly on the actomyosin complex (18)
. We therefore tested the effect of endogenous ADP accumulation near myosin ATPase on mitochondria-induced force modulation. Removal of the ATP-regenerating system by eliminating PCr, which leads to local accumulation of endogenous ADP (19)
, prevented the diminution of contractile force upon mitochondrial activation (Fig. 7
). With the addition of PCr, accumulation of ADP was dissipated by creatine kinase and the ability of mitochondrial activation to reduce force production was restored (Fig. 7A
). The same experiment performed in skinned fibers from creatine kinase null (CK–/–) mice, which lack all bound isoforms of creatine kinase (MM and mitochondrial), showed no force reduction with PCr addition, indicating that the effect of PCr was due specifically to ADP removal (Fig. 7B
). The sensitivity of isometric force production to mitochondrial energization could be partially restored in CK–/– mice by adding an exogenous ADP-eliminating system (Fig. 7C
).
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Finally, osmotic compression of the myofilament compartment in cardiac cells is known to reduce the length dependence of developed force (20)
. We caused such a compression by exposing skinned fibers to a solution containing 10% dextran. As shown in Fig. 8
A, B, osmotic compression considerably reduced the response of Ca2+-dependent and independent (rigor) force to mitochondrial energization. Changes in fiber configuration by variations in length also modulated the response to mitochondrial activation. All of our mechanical experiments were performed at sarcomere lengths of 
2.2 µm (20% above the slack length). Fiber shortening to the slack length (sarcomere length 1.85 µm) decreased the amplitude of force modulation after mitochondrial energization (Fig. 8C
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Many intracellular factors are known that can alter force development by cardiac cells and so may have mediated the effects of mitochondrial activation reported here. First, calcium uptake by energized mitochondria might cause a [Ca2+] decrease near the myofibrils, resulting in a decline in force. However, this is unlikely to have played a role for several reasons. Our experiments were performed in the presence of high Ca2+ buffer (EGTA). If mitochondria are able to locally decrease [Ca2+] near the myofibrils due to spatial interactions, then the force decline upon mitochondrial energization must be transitory because mitochondrial capacity to uptake calcium is not infinite. Furthermore, modulators of mitochondrial calcium uptake (ruthenium red and CGP37157) as well as thapsigargin, an inhibitor of sarcoplasmic reticulum ATPase, did not abolish the mechanical response to mitochondrial energization. In addition, mitochondrial functional state was able to modulate rigor tension development in the absence of calcium ions, indicating that calcium was not involved in this phenomenon.
Another intracellular metabolic factor controlling both maximal force development and calcium sensitivity of cardiac myofibrils is the ATP/ADP ratio (21
, 22)
. An increase in this ratio would decrease maximal force development as well as myofibrillar calcium sensitivity. Mitochondrial energization leading to ATP synthesis (and therefore ADP elimination) thus might be expected to decrease force development, even under conditions in which the intracellular medium composition is controlled due to direct energetic cross-talk between mitochondria and myofibrillar ATPase (7)
. However, this is unlikely to have mediated the effect reported here, because mitochondrial energization/de-energization was able to modulate rigor force, which is developed in the absence of adenine nucleotides. Moreover, we found that mitochondria could modulate force development in the presence of bongkrekic acid—that is, under conditions in which ADP rephosphorylation was blocked. These results also suggest that the mechanical response to mitochondrial energization is not related to local changes in inorganic phosphate, another negative modulator of myofibrillar function (23)
.
Finally, permanent proton production near the myofibrils could be responsible for force modulation after mitochondrial energization. Maximal force and calcium sensitivity of the myofibrils are both controlled by the cytosolic acid base equilibrium (24
25
26)
. Although the internal medium was strongly buffered with 60 mmol/L BES in our experiments, it is possible that mitochondrial energization activated a source of protons in the vicinity of the myofibrils that depressed force development. Protons are involved in the creatine kinase reaction, which occurs in the myofibrillar compartment and which may have been slowed by the activation of oxidative phosphorylation (7)
. However, the mechanical response to mitochondrial energization also occurred in the absence of adenine nucleotides (rigor tension), and thus in the absence of creatine kinase activity. On the other hand, the export of protons from the matrix upon energization could acidify the nearby myofibrillar compartment. However, nigericin, which abolishes the chemical proton gradient across the inner mitochondrial membrane, did not modify the force reduction caused by the mitochondrial energization.
Thus, it is unlikely that the chief regulators of myofibrillar function (Ca2+, ADP phosphorylation, pH) are involved in modulation of force by the mitochondrial functional state. Rather, the building or dissipation of m appears to play an important role in this process because treatment with FCCP reversed the mechanical response to mitochondrial energization. Similar results were obtained when the electrical component of m was removed using valinomycin but not after dissipation of the chemical (proton) component of m by nigericin. This would suggest either that the electrical potential has a direct effect on myofibrillar function or that m regulates a process that, in turn, influences the contractile apparatus. In the latter case, the most plausible explanation is that m regulates transmembrane ionic flux. Our experiments with various modulators of mitochondrial potassium flux favor this second hypothesis and suggest that m per se is not responsible for the mechanical response to mitochondrial energization. Indeed, our results establish that the mechanical response to mitochondrial activation/inactivation is mediated by transmembrane K+ fluxes.
How can potassium flux across the inner mitochondrial membrane modulate the force developed by the myofibrils? The main role attributed to transmembrane K+ flux is the regulation of mitochondrial matrix volume (14
, 27)
. Therefore, it is likely that the mechanism of force modulation involves changes in mitochondrial volume. To test this possibility, we monitored changes in mitochondrial morphology in situ using confocal microscopy and detecting the fluorescence derived from the membrane potential-insensitive dye MitoTracker Green. The main limitation of this approach is blurring of the fluorescent images, leading to overestimation of the mitochondrial volume fraction (10
, 28)
, but this does not impair the estimation of relative changes in mitochondrial volume. We found that all interventions resulting in force augmentation were associated with an increase in the area of MitoTracker Green fluorescence caused by swelling of the mitochondria.
Opinions differ on the effect of mitochondrial de-energization on mitochondrial volume. According to Garlid and Paucek (29)
, inhibition of the respiratory chain induces contraction of the mitochondrial matrix. However, experimental studies using cellular preparations show that mitochondria in situ undergo swelling under conditions of depolarization (30
, 31)
. In addition, results obtained in permeabilized cerebellar granule neurons by deconvolution microscopy demonstrate that de-energization with sodium azide or FCCP leads to a marked increase in mitochondrial volume (A. Kaasik, personal communication). Our results also agree with earlier studies showing that mitochondrial swelling is triggered by valinomycin (28
, 32)
or KATP channel openers (32
, 33)
. Matrix swelling in situ probably induces changes in the geometry of the whole mitochondrion, because an increase in matrix volume can trigger considerable changes in mitochondrial diameter even without stretching the outer membrane (31)
.
How changes in mitochondrial morphology influence the contractile apparatus has yet to be determined. The data presented here favor mechanical influence on the contractile apparatus. The characteristics of mitochondria-dependent force changes are similar to those observed when force development is modulated by cardiac cell length changes. First, mitochondrial de-energization increases force development at each [Ca2+] similar to the effect of fiber lengthening (17)
. Second, the mechanical response to mitochondrial energization disappears in the absence of an ATP-regenerating system, that is, under conditions favoring endogenous ADP accumulation in the myofibrillar compartment. Since this response occurs in the total absence of adenine nucleotides (rigor tension), it can be concluded that the absence of internal ADP rather than ATP replenishment is the prerequisite for this mechanical response. Again, this property resembles ADP-induced depression of the length dependence of force generation (18)
. Third, osmotic compression, which eliminates the length dependence of calcium sensitivity (20)
, markedly inhibits the mechanical response to mitochondrial energization. The precise mechanisms underlying the length dependence of force generation are not entirely known, but decreased intermyofilament spacing and/or changes in the strain of elastic proteins such as titin have been proposed (for a review, see ref 34
). It is therefore possible that mitochondrial geometry influences the contractile apparatus by mechanisms similar to those responsible for the length dependence of cardiomyocyte force production. The physiological role of mitochondrial volume regulation is poorly understood. It was proposed that control of matrix volume served to fine-tune the rate of oxidation by the respiratory chain (35)
or to ensure an optimal spatial arrangement of intermembrane proteins involved in energy transfer between mitochondria and the cytosol (16)
. Here, we suggest a new role for mitochondrial volume as a regulator of myofibrillar function. Furthermore, the data presented here suggest a new principle of mechanical signaling between intracellular compartments. The physical interaction of mitochondria with myofibrils could participate in the beat-to-beat regulation of force during shortening and lengthening of cardiac cells. The proposed role of mitochondria in the regulation of myofibrillar activity may have important pathophysiological implications. During energetic stress, for example, opening of K+ channels and/or blocking of the K+/H+ exchanger due to mitochondrial de-energization could increase the developed tension and consequently counteract the negative inotropic effect. Agents aimed at controlling transmembrane ionic fluxes through the mitochondrial membrane thus might provide valuable tools for modulating cardiac contractility.
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ACKNOWLEDGMENTS |
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Received for publication February 12, 2004. Accepted for publication April 21, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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  S. Belmonte and M. Morad 'Pressure-flow'-triggered intracellular Ca2+ transients in rat cardiac myocytes: possible mechanisms and role of mitochondria J. Physiol., March 1, 2008; 586(5): 1379 - 1397. [Abstract] [Full Text] [PDF]
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) or de-energized by 2 mM sodium azide (•). Experiments were performed in the presence of 25 µM bongkrekic acid to eliminate any ATP-generating mitochondrial activity. Force developed by fibers in the presence of azide at pCa 4.5 was taken as 100%.
