Metabolic consequences of functional complexes of mitochondria, myofibrils and sarcoplasmic reticulum in muscle cells
T. Andrienko1,2,
A. V. Kuznetsov1,3,
T. Kaambre4,
Y. Usson5,
A. Orosco1,
F. Appaix1,
T. Tiivel4,
P. Sikk4,
M. Vendelin6,
R. Margreiter3 and
V. A. Saks1,4,*
1 Laboratory of Fundamental and Applied Bioenergetics, INSERM E0221, Joseph
Fourier University, Grenoble, France
2 A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State
University, Moscow, Russia
3 Department of Transplant Surgery, University Hospital Innsbruck,
Innsbruck, Austria
4 Laboratory of Bioenergetics, National Institute of Chemical Physics and
Biophysics, Tallinn, Estonia
5 RFMQ-TIMC Laboratory, UMR 5525 CNRS, Institute Albert Bonniot, Grenoble,
France
6 Institute of Cybernetics, Tallinn, Estonia
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Fig. 1. Simultaneous imaging (co-localization) of mitochondrial flavoproteins (A)
and calcium (B) in permeabilized cardiomyocytes loaded with Rhod-2. (A)
Autofluorescence of mitochondrial flavoproteins in a fully oxidized state in
the absence of mitochondrial substrates shows a regular arrangement of
mitochondria in saponin-permeabilized cells. (B) Fluorescence of Rhod-2
trapped in mitochondria allows the localization and quantification of
mitochondrial free calcium. Clear co-localization of flavoproteins (green
fluorescence) and mitochondrial calcium (red fluorescence) can be seen.
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Fig. 2. Localization of cytoskeletal microtubular network in a selectively
permeabilized cardiomyocyte. Imaging of microtubular network by monoclonal
antibodies against tubulin demonstrates equal fluorescence over the entire
cardiomyocyte, indicating complete accessibility to these tubulin antibodies.
Cell size is the same as in Fig.
1.
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Fig. 3. Simultaneous imaging (co-localization) of mitochondrial flavoproteins (A)
and calcium (B) in permeabilized myocardial fibers loaded with Rhod-2. (A) As
in the case of isolated cells, autofluorescence of mitochondrial flavoproteins
in a fully oxidized state in the absence of mitochondrial substrates shows a
regular arrangement of mitochondria in saponin-permeabilized fibers. (B)
Fluorescence of Rhod-2 trapped in mitochondria allows the localization and
quantification of mitochondrial free calcium. As in cardiomyocytes
(Fig. 1), clear co-localization
of flavoproteins (green fluorescence) and mitochondrial calcium (red
fluorescence) can be seen.
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Fig. 4. Recordings of the respiration rate in permeabilized myocardial fibers
activated by endogenous ADP production in MgATPase reactions. Traces show the
rate of change of oxygen concentration in time in an oxygraph cell.
Respiration rates were measured in the presence of 5mmoll-1
glutamate plus 2mmoll-1 malate, as described in Materials and
methods. Addition of 2mmoll-1 ATP, and various final concentrations
of pyruvate kinase (PK) in the presence of 2mmoll-1
phosphoenolpyruvate (PEP) in the medium are indicated. At the end of
experiments, 20mmoll-1 creatine was added. Arrows show the time of
addition. The results show some inhibitory effect of the competitive pyruvate
kinase (PK)–PEP system for endogenous ADP on the respiration rate and a
stimulatory effect of creatine, due to coupled creatine kinase reaction, in
the presence of PK. Only minor inhibition of respiration by very high PK
activity (20i.u.ml-1) demonstrates compartmentation and direct
channeling of endogenous ADP. These effects of direct channeling are increased
after activation of mitochondrial creatine kinase.
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Fig. 6. Imaging of mitochondria in permeabilized myocardial fibers by the
membrane-potential-sensitive probe teramethylrhodamine ethyl ether (TMRE). (A)
Fibers in the presence of 2mmoll-1 ATP, 2mmoll-1 malate
and 5mmoll-1 glutamate (concentration of free Ca2+ in
Ca-EGTA buffer: 0.1 µmoll-1). (B) The same fibers after addition
of calcium chloride (final concentration of free Ca2+ in Ca-EGTA
buffer: 1.0 µmoll-1). The left fiber in a flexiperm chamber was
not fixed, while the right (longer) fiber was fixed by its ends. In A, both
fibers are relaxed. In B, the left fiber is contracted, while the right fiber,
which is contracting almost isometrically, shows significant structural
changes due to sarcomere contraction. Note the empty spaces between
mitochondria.
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Fig. 7. Imaging of mitochondria in permeabilized myocardial fibers after extraction
of myosin (ghost fibers) by membrane-potential-sensitive probe
teramethylrhodamine ethyl ether (TMRE). (A) Ghost fibers in the presence of
2mmoll-1 ATP, 2mmoll-1 malate and 5mmoll-1
glutamate (concentration of free Ca2+ in Ca-EGTA buffer: 0.1
µmoll-1). (B) The same ghost fibers after addition of calcium
chloride (final concentration of free Ca2+ in Ca-EGTA buffer: 1.0
µmoll-1). No structural changes were seen. The same result was
obtained for a free Ca2+ concentration of 3
µmoll-1.
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Fig. 8. Effect of different free Ca2+ concentrations on parameters of
ADP kinetics of mitochondrial respiration in permeabilized myocardial fibers
and in myosin-extracted (ghost) fibers. (A) Effect of Ca2+ on
apparent Km for ADP. A dramatic decline in the apparent
Km for ADP was observed in control fibers. By contrast, no
changes in apparent Km for ADP can be seen in ghost
fibers. (B) Effect of different free Ca2+ concentrations on
Vmax.
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Fig. 9. Scheme illustrating the functional intracellular energetic units (ICEUs) in
the cardiac cell. By interaction with cytoskeletal elements, the mitochondria
and sarcoplasmic reticulum (SR) are precisely fixed with respect to the
structure of the sarcomere of the myofibrils between two Z-lines and,
correspondingly, between two T-tubules. Calcium is released from the SR into
the space in the ICEU in the vicinity of the mitochondria and sarcomeres to
activate contraction and mitochondrial dehydrogenases. Adenine nucleotides
within the ICEU do not equilibrate rapidly with adenine nucleotides in the
bulk water phase. The mitochondria, SR and MgATPase of myofibrils and
ATP-sensitive systems in the sarcolemma are interconnected by metabolic
channeling of reaction intermediates and energy transfer within the ICEU by
the creatine kinase (CK)–phosphocreatine (PCr) and adenylate kinase (AK)
systems. CKcyt and AKcyt represent the CK and AK in the
cytoplasmic space. F0F1 is the mitochondrial ATPase
synthase complex. The protein factors (still unknown and marked as `X'), most
probably connected to cytoskeleton, fix the position of mitochondria and
probably also control the permeabilty of the VDAC channels to ADP and ATP.
Adenine nucleotides within the ICEU and bulk water phase may be connected by
some more rapidly diffusing metabolites than creatine (Cr)–PCr.
Synchronization of functioning of ICEUs within the cell may occur by the same
metabolites, for example, inorganic phosphate (Pi) or PCr, and/or
synchronized release of calcium during the excitation–contraction
coupling process. Adapted from Saks et
al., 2001 .
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© The Company of Biologists Ltd 2003
respiration rate before addition of ATP;