Intact mitochondria were isolated from heart and gastrocnemius muscle of WT and single- and double CK-knock-out mice strains cytosolic M-CK–⁄ –, mitochondrial Mi-CK–⁄ – and double knock-
Trang 1Mitochondrial affinity for ADP is twofold lower in creatine kinase knock-out muscles
Possible role in rescuing cellular energy homeostasis
Frank ter Veld1, Jeroen A L Jeneson2,3and Klaas Nicolay3
1 Department of Experimental In Vivo NMR, Image Sciences Institute, University Medical Center, Utrecht, the Netherlands
2 Department of Physiology, School of Veterinary Medicine, Utrecht University, the Netherlands
3 Biomedical NMR, Department of Biomedical Engineering, Eindhoven University of Technology, the Netherlands
Excitable mammalian cells contain high activities of
creatine kinase (CK, EC 2.7.3.2), which catalyses the
reversible exchange of a phosphoryl group between
phosphocreatine (PCr) and ATP The tissue-specific
CK enzymes are subcellularly compartmentalized and
consist of three cytosolic dimers: BB-CK (brain- and
smooth muscle-specific), MM-CK (muscle-specific)
and MB-CK heterodimers Furthermore, there is
mitochondrial CK (Mi-CK) which is located in the
intermembrane space of the mitochondrion and
con-sists mainly of octamers in vivo [1] Mi-CK and
M-CK have been hypothesized to jointly form an
energy transport network in which creatine (Cr) and PCr function as diffusible intermediates between sites
of ATP synthesis and utilization, thereby buffering fluctuations in the ATP free energy potential, i.e the ATP⁄ ADP concentration ratio [2,3] The roles
of Mi-CK and M-CK in this CK⁄ PCr shuttle model are to maintain a high local ADP⁄ ATP concentra-tion ratio near the adenine nucleotide translocase (ANT) by transphosphorylation of mitochondrially generated ATP to PCr and a high local ATP⁄ ADP ratio near extramitochondrial ATPases, respect-ively [4]
Keywords
heart; metabolic control; mitochondrial
respiration; skeletal muscle; transgenic mice
Correspondence
F ter Veld, Laboratory for Biophysics and
Cell Biology, Department of Epithelial Cell
Physiology, Max Planck Institute of
Molecular Physiology, Otto-Hahn-Strasse
11, D-44227 Dortmund, Germany
Fax: +49 231133 2299
Tel: +49 231133 2226
E-mail: frank.terveld@mpi-dortmund.mpg.de
(Received 30 July 2004, revised 8 December
2004, accepted 14 December 2004)
doi:10.1111/j.1742-4658.2004.04529.x
Adaptations of the kinetic properties of mitochondria in striated muscle lacking cytosolic (M) and⁄ or mitochondrial (Mi) creatine kinase (CK) iso-forms in comparison to wild-type (WT) were investigated in vitro Intact mitochondria were isolated from heart and gastrocnemius muscle of WT and single- and double CK-knock-out mice strains (cytosolic (M-CK–⁄ –), mitochondrial (Mi-CK–⁄ –) and double knock-out (MiM-CK–⁄ –), respect-ively) Maximal ADP-stimulated oxygen consumption flux (State3 Vmax; nmol O2Æmg mitochondrial protein)1Æmin)1) and ADP affinity (K ADP
50 ; lm) were determined by respirometry State 3 Vmax and K ADP
50 of M-CK–⁄ – and MiM-CK–⁄ – gastrocnemius mitochondria were twofold higher than those of WT, but were unchanged for Mi-CK–⁄ – For mutant cardiac mito-chondria, only the K50ADP of mitochondria isolated from the MiM-CK–⁄ – phenotype was different (i.e twofold higher) than that of WT The implica-tions of these adaptaimplica-tions for striated muscle function were explored by constructing force-flow relations of skeletal muscle respiration It was found that the identified shift in affinity towards higher ADP concentra-tions in MiM-CK–⁄ –muscle genotypes may contribute to linear mitochond-rial control of the reduced cytosolic ATP free energy potentials in these phenotypes
Abbreviations
ACR, acceptor control ratio; AT, atractyloside; CK, creatine kinase; Cr, creatine; CS, citrate synthase; EDL, extensor digitorum longus; FCCP, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone; LDH, lactate dehydrogenase; PCr, phosphocreatine; RCR, respiratory control ratio; VDAC, voltage-dependent anion channel.
Trang 2Loss of CK function either by depletion of Cr via
beta-guanidinopropionic acid feeding [5,6] or by
dele-tion of CK isoforms in striated muscle weakens
con-trol of ATP⁄ ADP concentration ratios in the cellular
ATPase network [7–9] Elevated ADP concentrations
compared to wild-type (WT) have been measured at
steady states set by comparable cytosolic ATPase rates
in Mi-CK knockout hearts [8,9] and M-CK knockout
fast-twitch gastrocnemius muscle [7] compared to WT
In the latter muscle type, this is the case both at rest
as well as during contraction, in spite of phenotypic
adaptations of the muscle at the protein level For
example, a shift in the myosin composition of the
myofibrils towards slower, energetically more efficient
isoforms has been documented for fast-twitch muscle
in response to CK deletion [10]
The adaptive response of mitochondrial function in
CK-deficient muscle cells is less well documented
Deletion of CK function leads to increased citrate
syn-thase (CS) activity in skeletal muscle and an increased
Vmax of ADP-stimulated respiration in gastrocnemius
skinned-fibres [11] Here we investigated if the ADP
concentration increase found in CK-deficient muscle is
accompanied by a compensatory, adaptive shift in
mito-chondrial ADP affinity towards these higher ADP
concentrations We measured the ADP-stimulated
Vmax of respiration and the affinity for ADP (K50ADP)
in isolated mitochondria from two extreme striated
muscle phenotypes: slow-twitch heart and fast-twitch
gastrocnemius muscle
Results
Isolation of mouse heart and gastrocnemius
mitochondria
Percoll density gradient centrifugation was added as a
final purification step to obtain a high quality
mitoch-ondrial preparation CS activity was increased in Percoll
purified mitochondria when compared to the heart
homogenate and the crude mitochondrial preparation,
albeit not significantly (Table 1) Based on the activity
of aryl esterase (AE) as a microsomal marker, 7% of the
microsomal contamination remained in the final
mito-chondrial preparation (on protein basis) when compared
to the homogenate (Table 1) The final mitochondrial suspension was furthermore greatly deprived of lactate dehydrogenase (LDH) activity, as a cytosolic marker One of the most important quality criteria for the final mitochondrial preparation is the respiratory control ratio (RCR) The crude mitochondrial fraction had a low RCR (2.6 ± 0.3) and a relatively high ATPase activity (Table 1) Considerable levels of contaminating ATPases remained in the final mitochondrial sample during isolation of mouse heart mitochondria when con-ventional differential centrifugation protocols were used (Table 1) The reduction of the ATPase activity in the final heart mitochondrial preparation was accompanied
by a considerably higher RCR of 11.2 ± 1.7, using pyruvate⁄ malate as substrate (Table 1) Percoll density gradient centrifugation also strongly increased the RCR
of the gastrocnemius mitochondrial preparation, i.e from 1.9 ± 0.4 to 5.9 ± 0.5, using succinate as sub-strate (data not shown)
Creatine kinase activity Table 2 shows the specific activity of CK in mouse heart and gastrocnemius homogenates as well as in mitochondria isolated from these WT and CK-deficient mouse tissues In agreement with the genotypes, the
CK activities in mitochondria isolated from Mi-CK–⁄ – and MiM-CK–⁄ – mouse heart were negligible Import-antly, the data show that there is no significant change
in Mi-CK activity in the case of M-CK deficiency The total CK activity was significantly lower in the heart homogenate of the three CK-deficient mice com-pared to WT mice In the gastrocnemius homogenate the total CK activities of WT and Mi-CK–⁄ – were not significantly different, which is in line with the low Mi-CK content in glycolytic gastrocnemius muscle Isolated gastrocnemius mitochondria from WT and M-CK–⁄ – mice displayed a relatively low specific Mi-CK activity, compared to heart mitochondria In preparations of mitochondria isolated from Mi-CK–⁄ – gastrocnemius the relatively high CK activity, com-pared to mitochondria from MiM-CK–⁄ – muscle, is probably due to contamination with M-CK
Table 1 RCR, ATPase activity and marker enzyme activities for mouse heart homogenate, crude mitochondria and purified mitochondria Number of preparations is shown in parentheses Activities are shown for ATPase, CS, LDH and AE (mUÆmg protein)1).
Trang 3Vmaxof heart and gastrocnemius mitochondrial
respiration
The basic respiratory rates for maximal ADP
stimula-ted (State 3), the atractyloside inhibistimula-ted (AT) state and
the optimally uncoupled (FCCP) state were essentially
identical across the different types of cardiac
mito-chondria (Table 3, A) Interestingly, respiratory rates
in State 3, AT state and FCCP state were significantly
higher in isolated gastrocnemius mitochondria from
M-CK–⁄ – and MiM-CK–⁄ – mice, compared to WT
(Table 3, B) The respiratory rates of isolated
gastroc-nemius mitochondria from WT and Mi-CK-deficient
mice were not significantly different An acceptor
control ratio (ACR), and not an RCR, was calculated from ADP titration experiments using State 3 and AT state rates due to the limited amount of mitochondria obtained from gastrocnemius muscle
K50ADP of heart and gastrocnemius mitochondrial respiration
In the presence of Cr, the concentration of ADP nee-ded to induce half-maximal respiration in isolated car-diac mitochondria, the apparent K50 value for ADP (K50ADP), was expectedly and significantly lowered from 21.3 ± 2.8 lm to 15.8 ± 1.6 lm and from 20.5 ± 1.7 lm to 14.5 ± 0.2 lm for mitochondria from WT and M-CK–⁄ – myocardium, respectively (Table 3, A) For heart mitochondria from Mi-CK–⁄ – and MiM-CK–⁄ – mice, these values, in the presence of
Cr, were 21.0 ± 4.7 lm and 32.2 ± 4.2 lm, respect-ively, and did not differ when Cr was omitted (Table 3, A) As such, the K ADP
50 in the presence of
Cr, representative of the conditions in vivo, of heart mitochondria was twofold higher for MiM-CK–⁄ –mice (P < 0.05) and tended to be higher (1.3-fold; not sig-nificant) for Mi-CK–⁄ –mice compared to WT
The apparent K50 for ADP of gastrocnemius muscle mitochondria, in the absence of Cr, were 7.0 ± 1.0 lm and 7.3 ± 1.0 lm for M-CK–⁄ – and MiM-CK–⁄ –, respectively, vs 2.4 ± 0.3 lm for WT, and 6.4 ± 0.8 lm and 5.7 ± 0.7 lm vs 3.5 ± 0.3 lm, respectively, when Cr was present (Table 3, B) No differences were found between WT and Mi-CK–⁄ – gastrocnemius mitochondria (Table 3, B) K ADP
in all cases lower than for cardiac mitochondria
Table 2 CK activities in muscle homogenates and isolated
mito-chondria from WT and CK-deficient mice Number of preparations
is shown in parentheses.
Creatine kinase activity (nmol ADPÆmg protein)1Æmin)1)
Mi-CK – ⁄ –
MiM-CK – ⁄ –
*P < 0.05 compared to WT.
Table 3 Kinetic characterization of succinate ⁄ rotenone-dependent respiration of isolated heart and gastrocnemius mitochondria from WT and CK-deficient mice Respiratory rates of isolated mouse heart (A) and gastrocnemius (B) mitochondria (0.1 mgÆmL)1) were measured in mitochondrial medium (see Experimental procedures) containing succinate as substrate and rotenone The RCR value (A) is the ratio of state
3 over state 4 (data not shown) The ACR value (B) is the ratio of state 3 over the atractyloside-inhibited state For the determination of apparent K 50 steady-state respiratory rates were measured at increasing [ADP] Mi-CK activity was induced by adding 25 m M Cr Number of experiments is shown in parentheses.
Respiratory Rate (nmol O2Æmg mitochondrial protein)1Æmin)1)
RCR (ACR)
App K50for ADP (l M )
MiM-CK – ⁄ –
M-CK – ⁄ –
*P < 0.05 compared to WT **P < 0.05 compared to minus Cr (–Cr).
Trang 4(Table 3, B) In addition, the sensitivity of WT
mito-chondria to the presence of Cr in the medium differed
between gastrocnemius and cardiac preparations:
addi-tion of Cr to the medium significantly increased
K ADP
(Table 3, B) In contrast, the K ADP
50 of gastrocnemius mitochondria from M-CK–⁄ – and MiM-CK–⁄ – mice
was not sensitive to the presence of Cr in the medium,
and was significantly higher than WT in both
condi-tions studied
Discussion
In this study we compared the functional kinetic
char-acteristics of mitochondria from WT and CK-deficient
mice in fast-twitch gastrocnemius and slow-twitch
heart muscle, which represent two very different
stri-ated muscle phenotypes
Fast-twitch glycolytic skeletal muscle
The main finding of our studies on mitochondria
isola-ted from various CK genotypes of fast-twitch
gastroc-nemius muscles was a twofold higher rate of
endogenous and State3 respiration (Vmax) and a
two-fold higher apparent K50 for ADP for M-CK–⁄ – and
MiM-CK–⁄ – mice compared to WT mitochondria
(Table 3, B) Mitochondria from Mi-CK–⁄ –
gastro-cnemius had essentially the same respiratory properties
as WT mitochondria, being in line with previous
reports [12] (Table 3, B) The finding of an adaptive
increase in respiratory Vmax in M-CK–⁄ – and
MiM-CK–⁄ – gastrocnemius mitochondria is in line with the
results of previous studies on muscle homogenate that
reported an increase of mitochondrial protein in these
genotypes [10,13–15] Also, the results of polarographic
measurements of respiratory Vmax (but not K ADP
50 ; see [16]) in permeabilized M-CK–⁄ – gastrocnemius fibres,
which can be compared to our results in a
straightfor-ward manner, are similar [11,17] Our present
investi-gations did not provide insight into the exact sites of
Vmax up-regulation in M-CK–⁄ – and MiM-CK–⁄ –
phe-notypes in terms of activities of individual components
of the respiratory chain However, an interesting, but
speculative, scenario could be that the documented
cal-cium homeostasis impairment due to CK deficiency
[18], possibly resulting from loss of CK function [19],
may have played a role in directing the increase in
mitochondrial capacity via the recently discovered
cal-modulin-kinase calcium-signalling pathway controlling
mitochondrial biogenesis [20]
The K ADP
50 in the presence of Cr, representative of
the conditions in living muscle, was 6.4 lm and 5.7 lm
for M-CK–⁄ – and MiM-CK–⁄ –, respectively, compared
to 3.5 lm for WT gastrocnemius mitochondria This twofold-decrease in affinity for ADP in these two phenotypes is physiologically relevant in view of the reported twofold higher ADP concentration in resting MiM-CK–⁄ – hindleg muscles [7] as will be discussed below The apparent K ADP
50 is determined by the per-meability of the outer mitochondrial membrane to ADP via VDAC porins [21] and the affinities of ANT and F1-ATPase for ADP [22] The latter also introduces
a dependence on the mitochondrial membrane potential and thereby on the respiratory substrate [23] The poss-ible role of mitochondrial adenylate kinase in setting the apparent K50ADP was not addressed in this study However, the Vmaxactivities of mitochondrial adenylate kinase in isolated heart or gastrocnemius mitochondria from CK-deficient genotypes were not significantly dif-ferent compared to WT mitochondria (data not shown) This makes it unlikely that adenylate kinase is the source of the observed differences in K ADP
Interestingly, recent experimental data reveal a relat-ive decrease in VDAC mRNA and protein expression compared to the expression of other mitochondrial proteins in M-CK–⁄ – and MiM-CK–⁄ – gastrocnemius muscle [13,24] suggesting a lower permeability of the outer mitochondrial membrane for adenine nucleotides The decrease in ADP affinity of isolated M-CK–⁄ – and MiM-CK–⁄ – muscle mitochondria we found is therefore in line with these findings at the protein level In addition, experiments on VDAC-1 deficient mouse gastrocnemius have clearly shown VDAC to be a important determinant in setting
K ADP
50 , giving rise to twofold higher K ADP
upon VDAC-1 deletion [25]
Slow-twitch oxidative cardiac muscle
No significant differences in mitochondrial respiratory
Vmax were found when comparing isolated mitochon-dria from heart muscle from Mi-CK–⁄ –, M-CK–⁄ – and MiM-CK–⁄ – mice with heart mitochondria from WT mice (Table 3, A) These findings are in line with previous studies on skinned ventricular fibres from Mi-CK–⁄ – and M-CK–⁄ – mice that reported no differ-ence in respiratory Vmax compared to WT [11,12] Our finding of twofold higher K ADP
50 of MiM-CK–⁄ – heart mitochondria and the trend towards a higher K ADP
the case of Mi-CK–⁄ –mitochondria correlates well with recent studies on perfused hearts from CK mutant ani-mals In these studies a compromised capacity for free energy homeostasis was demonstrated in isolated per-fused heart from Mi-CK–⁄ –and MiM-CK–⁄ –mice [8,9], but not M-CK–⁄ –mice [8,26]
Trang 5Integration of adapted mitochondrial function
in the CK mutant striated muscle cell
In this section we discuss the implications of the
identi-fied Vmax and K ADP
50 adaptations of mitochondria with respect to the function of the integrated ATPase
network of the active striated muscle cell in which
spe-cific CK isoforms are absent The role of mitochondria
in the ATPase network of the cell is to both generate
ATP synthase flux matching cytosolic ATPase flux as
well as to control the extramitochondrial ATP⁄ ADP
free energy potential [27] This is captured in Fig 1
which shows respiratory flux of WT muscle as a
func-tion of the extramitochondrial ATP⁄ ADP free energy
potential This relationship is quasi-linear over 5–85%
of respiratory Vmax in skeletal muscle [28], with the
operational ATP synthase flux domain being able to
maintain adequate control over cytosolic ATP⁄ ADP [27,28] Above this maximal operational ATP synthase rate, respiration can no longer control cytosolic ATP⁄ ADP and the free energy potential rapidly deteri-orates
The kinetic graph format of Fig 1 will now be used
to qualitatively illustrate (i.e focusing on trends rather than absolute numbers) the implications of the mitoch-ondrial Vmax and K50ADP adaptations to (Mi)M-CK-deficient skeletal muscle physiology In order to do so,
we first translated the relative change in K50ADP to
in vivo conditions on basis of information in the litera-ture This was necessary because K50ADP values for iso-lated mitochondria are typically lower than estimated
in vivo values {5 lm (this study) vs 23–44 lm [28–30], respectively, for skeletal muscle, and 20–30 lm [31,32]
vs 80 lm [33], respectively, in cardiac muscle oxidizing glucose} For skeletal muscle, we thus obtained an
in vivoK ADP
50 of 72 lm for MiM-CK-deficient skeletal muscle on basis of an in vivo K ADP
skeletal muscle of 44 lm [29] and the 1.6-fold increase
in in vitro K ADP
50 for MiM-CK–⁄ – compared to WT (Table 3, B) These translated K ADP
50 values together with measured in vitro mitochondrial Vmax rates were first converted to muscle Vmax rates assuming 10.3 mg mitochondrial proteinÆg skeletal muscle tissue mass)1 [34] and then used to construct flow-force relations for three cases: (I) WT muscle characterized by Vmax¼ (Vmax)WT and K ADP
50 )WT; (II) MiM-CK–⁄ – characterized by Vmax¼ 2*(Vmax)WT and K ADP
2*(K ADP
50 )WT; (III) a hypothetical case characterized
by Vmax¼ 2*(Vmax)WT and K ADP
50 )WT (Fig 1) In the final step, we calculated the ATP⁄ ADP free energy potential in resting WT and MiM-CK defi-cient fast-twitch mouse extensor digitorum longus (EDL) muscle on the basis of reported PCr, Cr and ATP concentrations at 20C [35] and a value of 166 for CK-Keq [36] yielding ATP⁄ ADP ratios of 533 and
163 for WT and MiM-CK–⁄ – EDL, respectively This approach was valid because at rest thermodynamic equilibrium is also established in MiM-CK–⁄ – due to the presence of some remaining CK activity [18] The free energy offset-points of the ATPase network for the two genotypes are indicated in Fig 1 by broken lines Clearly, the cytosolic ATP free energy potential
in MiM-CK–⁄ – fast-twitch muscle is compromised already under conditions of basal ATP demand The flow–force relationship for WT muscle first of all shows that without any adaptation of Vmax or
50 , mitochondria in MiM-CK-deficient skeletal muscle would have a seriously compromised dynamic range to respond to cytosolic ATPase load increments This is because the ATP⁄ ADP free energy potential
Fig 1 Qualitative illustration of flow-force relations in fast-twitch
skeletal muscle of WT and MiM-CK-deficient mice
Extramitochon-drial ATP free energy potential represented by the ATP ⁄ ADP ratio
in skeletal muscle from WT and MiM-CK – ⁄ – mice is plotted against
muscle respiratory flux (JO2in nmoles O2Æg muscle -1 Æmin -1 ), based
on converted mitochondrial respiratory V max rates and extrapolated
K 50 values from Table 3B (in the presence of Cr) Three cases are
presented: (I) WT with Vmax¼ (V max )WT and K ADP
50 ¼ (K ADP
50 )WT; (II) MiM-CK – ⁄ –
with V max ¼ 2*(V max ) WT and K50ADP¼ 2*(K 50ADP) WT ;
and (III) a hypothetical case with V max ¼ 2*(V max ) WT and K50ADP¼
(K ADP
50 )WT The free energy ATP ⁄ ADP offset-points at rest of the
ATPase network for the two genotypes (case I and II) are indicated
by dashed lines The arrows indicate the available dynamic range to
respond to cellular ATPase load increments [with the WT (arrow A)
and compromised MiM-CK – ⁄ –
(arrows B, C and D) free energy ATP ⁄ ADP potential as offset-point] The gray boxes indicate the
quasi-linear domains of respiratory Vmax.
Trang 6offset-point has shifted in MiM-CK-deficient muscle
from 533 to 163, giving rise to an increase in basal
res-piratory rate from 25% to 60% WT Vmax (Fig 1, case
I, arrows A and B, respectively) This would pose a
problem, as the absolute cytosolic ATPase load during
contraction in MiM-CK-deficient muscle is higher than
for WT because of an increased basal rate associated
with the compromised Ca2+ homeostasis, as observed
in CK deficiency [18] In addition, we recently obtained
experimental proof for higher absolute respiration
rates in MiM-CK-deficient mouse EDL muscles at one
and the same contraction frequency compared to WT
due to a significantly increased basal respiration rate
(F ter Veld, unpublished data) Secondly, the
relation-ship for MiM-CK-deficient skeletal muscle (case II)
shows that the increase in respiratory Vmax of
mito-chondria in this genotype rescues the absolute capacity
to generate ATP synthase flux, as compared to
mito-chondria with WT Vmax (clearly illustrated by
com-paring the dynamic range of arrows C and B,
respectively) In addition, the observed increase in
K ADP
50 shifts the dynamic range of ATP synthase flux
in MiM-CK-deficient muscle (arrow C) to a more
lin-ear range of respiratory flux (grey box, case II),
com-pared to rather small linear range (grey box, case III)
corresponding to the dynamic range in case III (arrow
D) This hypothetical case III illustrates the
import-ance of combining these two kinetic properties, in that
while an increase of Vmax may be essential to restore
one aspect of mitochondrial function, i.e ATP
syn-thase flux, a second crucial aspect has to be
main-tained in addition, i.e control of the cytosolic ATP
free energy potential This second aspect is resolved by
an adaptive response of a twofold higher K ADP
MiM-CK-deficient muscle In this light, it is of interest
that a doubling of K ADP
50 has also been found in skel-etal muscle of patients with mitochondrial lesions
reducing Vmax by 50% [37,38] In spite of the severely
reduced capacity to generate ATP synthase flux, these
muscles have residual capacity for contractile work
accompanied by linear changes in cytosolic ATP free
energy at low ATP⁄ ADP potentials [37,38]
Analogously, we can now explain the benefit of an
increased mitochondrial K ADP
50 in MiM-CK–⁄ – hearts
in which mitochondrial control of the cytosolic ATP
free energy potential is compromised [8,26] One would
perhaps have expected also a higher mitochondrial Vmax
in these cardiac muscle genotypes An attractive, but
speculative, explanation for the lack of any such Vmax
increase is offered by Lindstedt et al [39] who have
pro-posed that the volume ratio of mitochondria,
sarcoplas-mic reticulum and myofibrils in a striated muscle cell is
optimized for the particular mechanical task of the
muscle Our results suggest that cardiac muscle may well be limited in its ability to increase mitochondrial volume without compromising mechanical function, at least in comparison to fast-twitch skeletal muscle
In conclusion, we propose that an increase in oxida-tive capacity and a reduction of the ADP affinity both constitute adaptations of mitochondrial function to alleviate compromised temporal and spatial buffering
of the ATP free energy potential due to specific CK deletions A specific mechanism for the regulation of mitochondrial capacity has recently been identified [20] It remains to be determined which regulatory mechanisms are involved in setting the apparent mito-chondrial K ADP
Experimental procedures
Animals
Adult WT C57BL⁄ 6 mice were used as controls Cytosolic muscle-type CK-deficient mice (M-CK– ⁄ –), sarcomeric mit-ochondrial CK-deficient mice (Mi-CK–⁄ –) and double knock-out mice, deficient in both cytosolic muscle-type and sarcomeric mitochondrial CK (MiM-CK–⁄ –), were gener-ated in the laboratory of B Wieringa (Nijmegen University, the Netherlands) by gene targeting as described previously [10,15] Offspring obtained in the breeding program were genotyped by PCR analysis on a regular basis All experi-mental procedures were approved by the Committee on Animal Experiments of the University Medical Center Utrecht and complied with the principles of good laborat-ory animal care
Biochemicals
Percoll was from Pharmacia Biotech (Rosendaal, the Netherlands) Essentially fatty acid free BSA, lyophilized Leuconostoc mesenteroides glucose-6-phosphate dehydroge-nase (NAD+ specific form) and lyophilized yeast HK (essentially salt free) were from Sigma (Zwijndrecht, the Netherlands) ATP and ADP were obtained from Roche Diagnostics (Almere, the Netherlands) All other chemicals used were of the highest grade available and were obtained from regular commercial sources
Preparation of heart muscle mitochondria
The isolation of mitochondria from mouse heart was based
on the procedure of Cairns et al [40], which represents a modification of the technique of Sims [41] For each prepar-ation, four mice were sedated with diethyl-ether and decap-itated after which beating hearts were removed The hearts (approx 500 mg total wet-weight) were quickly placed in isolation medium [IM, containing 250 mm mannitol, 10 mm
Trang 7Hepes, 0.5 mm EGTA and 0.1% (w⁄ v) BSA, pH 7.4;
adjus-ted with KOH] Next, the ventricles were carefully freed of
blood, minced intensively in 5 mL IM using scissors and
homogenized in a 12 mL centrifuge tube by five strokes (up
and down) using a loosely fitting Teflon pestle rotating at
1000 r.p.m Large cell debris and nuclei were pelleted by
centrifugation for 5 min at 500 g in a Sorvall SS34 rotor
Mitochondria were pelleted by centrifuging the supernatant
for 5 min at 10 000 g in the same rotor The mitochondrial
pellet was resuspended in 2 mL 12% (v⁄ v) Percoll in IM,
loaded on a discontinuous density gradient consisting of
3 mL 26% (v⁄ v) Percoll and 4 mL 40% (v ⁄ v) Percoll in IM
and centrifuged for 5 min at 31 000 g in a Sorvall SS34
rotor Three major bands were obtained and the purified
mitochondria were collected from the bottom band
contain-ing high-density mitochondria Finally, the mitochondria
were washed with IM by centrifuging twice for 5 min at
10 000 g and resuspended in 200 lL IM at a mitochondrial
protein concentration of 12 mgÆmL)1 The isolations
typ-ically took 45 min and were carried out at a temperature of
0–4C
Preparation of gastrocnemius muscle
mitochondria
The isolation of mitochondria from mouse gastrocnemius
was essentially the same as procedure described above for
heart mitochondria, with some minor modifications Four
mice were sedated with diethyl-ether and decapitated after
which hindleg gastrocnemius muscles were removed, placed
in IM and freed of fat tissue The muscle tissue was minced
intensively in IM using scissors and homogenized in a
cen-trifuge tube by five strokes (up and down) using a loosely
fitting Teflon pestle rotating at 700 r.p.m To obtain
gas-trocnemius mitochondria, again a discontinuous density
gradient was used The 26% (v⁄ v) Percoll layer was
replaced with a 20% (v⁄ v) Percoll layer Two major bands
were obtained and the purified mitochondria were collected
from the bottom band containing high-density
mitochon-dria Finally, the mitochondria were washed with IM as
described above and resuspended in IM to a mitochondrial
protein concentration of approximately 5 mgÆmL)1
Protein determination
The protein concentration of the mitochondrial preparation
was determined by the BCA assay (Pierce, Etten-Leur, the
Netherlands) The BCA reagent was supplemented with
0.1% (w⁄ v) SDS BSA was used as standard
Measurements of respiratory parameters
The rates of oxygen consumption (nmol O2Æmg
mitochond-rial protein)1Æmin)1) were determined at 25C, using a
high-resolution oxygraph (Oroboros Oxygraph; Innsbruck, Austria) and 0.1 mg mitochondria in mitochondrial med-ium [containing 200 mm sucrose, 20 mm Hepes, 20 mm tau-rine, 10 mm KH2PO4, 3 mm MgCl2, 0.5 mm EGTA, 0.1% (w⁄ v) BSA, pH 7.4 adjusted with KOH] The final volume
of the oxygraph chamber was 2.0 mL The oxygen solubil-ity of air-saturated mitochondrial medium was taken to be
221 nmol O2ÆmL)1 [42] Substrates were 10 mm pyruvate plus 2 mm malate, or 10 mm succinate (in the presence of
10 lm rotenone) Respiratory assays were typically carried out in the following order Endogenous respiration (State 2) was measured before the submaximal stimulation of oxida-tive phosphorylation using 0.1 mm ADP while maximal ADP stimulated respiration (State 3) was initiated by add-ing 0.25 mm ADP After the restadd-ing state (State 4) had again been reached, 12.5 lm atractyloside was added to measure the rate of ANT-inhibited respiration Finally, approximately 2 lm FCCP was titrated into the oxygraph chamber to induce maximally uncoupled respiration The apparent K50 values for ADP, i.e., the concentration of ADP needed to induce half-maximal respiration in isolated mitochondria, were determined by measuring respiration at increasing [ADP] in mitochondrial medium containing
10 mm succinate, 10 lm rotenone, 20 mm glucose and 0.3 IUÆmL)1 yeast hexokinase (type VI), for depletion of mitochondrially formed ATP The ADP concentration of stock solutions was determined enzymatically as described before [21] To assess functional coupling of Mi-CK to oxi-dative phosphorylation, respiration was stimulated at increasing [ADP] in the presence of 25 mm Cr To obtain the rate of ADP-stimulated respiration, the rates of respir-ation were corrected for ‘leak’ respirrespir-ation based on a dynamic computer model of oxidative phosphorylation in muscle [43] according to [44]
Spectrophotometric determination of enzyme activities
CK activity was measured at 25C on a Beckman DU65 spectrophotometer using coupled enzyme systems Briefly,
CK activity was assayed according to [45] in the forward direction in a medium containing 10 mm imidazole, 2 mm EDTA, 10 mm Mg-acetate, 2 mm ADP, 20 mm N-acetyl-cysteine, 20 mm glucose, 5 mm AMP, 1 mm NAD+, 50 lm
P1, P5-di(adenosine-5¢)pentaphosphate, 25 mm PCr (pH 7.4, adjusted with acetic acid) Hexokinase and glucose-6-phos-phate dehydrogenase were added at 3 IUÆmL)1 and
2 IUÆmL)1, respectively Pyruvate kinase and lactate dehy-drogenase were both added at 4.5 IUÆmL)1 Lactate dehy-drogenase [46], citrate synthase [47] and aryl esterase [48] enzyme activities were measured at 37C and pH 7.4 according to published methods The media used in the above assays were adjusted to 0.2% Triton X-100 to obtain maximal enzyme activities in muscle homogenates and
Trang 8mitochondrial fractions Total ATPase activities in
suspen-sions of intact mitochondria were measured as described
previously [46,49] Care was taken to avoid detergent
con-tamination and no Triton X-100 was added
Data analysis and statistics
Oxygraph data analysis was performed with high-resolution
respirometry software (oroboros datlab 2.1; Innsbruck,
Austria) Apparent K ADP
50 values were calculated using nonlinear regression (kaleidagraph 3.0, Synergy Software,
Reading, USA) assuming second-order Hill kinetics [28]
Reported data are presented as arithmetic means ± SEM
Statistical analyses were performed using Student’s t-test
Differences between means were considered significant if
P< 0.05
Acknowledgements
We thank F.N Gellerich, E Gnaiger, B de Kruijff
and B Wieringa for expert advice We thank B
Wier-inga, F Oerlemans and K Steeghs (Nijmegen
Univer-sity) for supplying the transgenic mice This research
was supported by The Council for Chemical Sciences
of the Netherlands Organization for Scientific
Research (CW-NWO)
References
1 Brdiczka D, Kaldis P & Wallimann T (1994) In vitro
complex formation between the octamer of
mitochon-drial creatine kinase and porin J Biol Chem 269,
27640–27644
2 Wallimann T, Wyss M, Brdiczka D, Nicolay K &
Eppenberger HM (1992) Intracellular
compartmenta-tion, structure and function of creatine kinase
iso-enzymes in tissues with high and fluctuating energy
demands: the ‘phosphocreatine circuit’ for cellular
energy homeostasis Biochem J 281, 21–40
3 Wyss M, Smeitink J, Wevers RA & Wallimann T (1992)
Mitochondrial creatine kinase: a key enzyme of aerobic
energy metabolism Biochim Biophys Acta 1102,
119–166
4 Saks VA, Kuznetsov AV, Kupriyanov VV, Miceli MV
& Jacobus WE (1985) Creatine kinase of rat heart
mito-chondria The demonstration of functional coupling to
oxidative phosphorylation in an inner membrane-matrix
preparation J Biol Chem 260, 7757–7764
5 Moerland TS & Kushmerick MJ (1994) Contractile
economy and aerobic recovery metabolism in skeletal
muscle adapted to creatine depletion Am J Physiol 267,
127–137
6 Wiseman RW & Kushmerick MJ (1995) Creatine kinase
equilibration follows solution thermodynamics in
skele-tal muscle 31P NMR studies using creatine analogs
J Biol Chem 270, 12428–12438
7 Gorselink M, Drost MR, Coumans WA, van Kranen-burg GP, Hesselink RP & van der Vusse GJ (2001) Impaired muscular contractile performance and adenine nucleotide handling in creatine kinase-deficient mice
Am J Physiol Endocrinol Metab 281, 619–625
8 Saupe KW, Spindler M, Hopkins JC, Shen W & Ing-wall JS (2000) Kinetic, thermodynamic, and develop-mental consequences of deleting creatine kinase isoenzymes from the heart Reaction kinetics of the creatine kinase isoenzymes in the intact heart J Biol Chem 275, 19742–19746
9 Spindler M, Niebler R, Remkes H, Horn M, Lanz T & Neubauer S (2002) Mitochondrial creatine kinase is cri-tically necessary for normal myocardial high-energy phosphate metabolism Am J Physiol Heart Circ Physiol
283, 680–687
10 Steeghs K, Heerschap A, de Haan A, Ruitenbeek W, Oerlemans F, van Deursen J, Perryman B, Pette D, Bruckwilder M, Koudijs J, Jap P & Wieringa B (1997) Use of gene targeting for compromising energy homeos-tasis in neuro-muscular tissues: the role of sarcomeric mitochondrial creatine kinase J Neurosci Methods 71, 29–41
11 Veksler VI, Kuznetsov AV, Anflous K, Mateo P, van Deursen J, Wieringa B & Ventura-Clapier R (1995) Muscle creatine kinase-deficient mice II Cardiac and skeletal muscles exhibit tissue-specific adaptation of the mitochondrial function J Biol Chem 270, 19921–19929
12 Boehm E, Veksler V, Mateo P, Lenoble C, Wieringa B
& Ventura-Clapier R (1998) Maintained coupling of oxidative phosphorylation to creatine kinase activity in sarcomeric mitochondrial creatine kinase-deficient mice
J Mol Cell Cardiol 30, 901–912
13 de Groof AJ, Oerlemans FT, Jost CR & Wieringa B (2001) Changes in glycolytic network and mitochondrial design in creatine kinase-deficient muscles Muscle Nerve
24, 1188–1196
14 Tullson PC, Rush JW, Wieringa B & Terjung RL (1998) Alterations in AMP deaminase activity and kinetics in skeletal muscle of creatine kinase-deficient mice Am J Physiol 274, 1411–1416
15 van Deursen J, Heerschap A, Oerlemans F, Ruitenbeek
W, Jap P, ter Laak H & Wieringa B (1993) Skeletal muscles of mice deficient in muscle creatine kinase lack burst activity Cell 74, 621–631
16 Kongas O, Wagner MJ, ter Veld F, Nicolay K, van Beek JHGM & Krab K (2004) The mitochondrial outer membrane is not a major diffusion barrier for ADP
in mouse heart skinned fibre bundles Pflugers Arch-European J Physiol 447, 840–844
17 Kaasik A, Veksler V, Boehm E, Nototova M & Ventura-Clapier R (2003) From energy store to energy
Trang 9flux: a study in creatine kinase deficient fast skeletal
muscle FASEB J 17, 708–710
18 Steeghs K, Benders A, Oerlemans F, de Haan A,
Heers-chap A, Ruitenbeek W, Jost C, van Deursen J,
Perry-man B, Pette D, Bruckwilder M, Koudijs J, Jap P,
Veerkamp J & Wieringa B (1997) Altered Ca2+
responses in muscles with combined mitochondrial and
cytosolic creatine kinase deficiencies Cell 89, 93–103
19 Korge P & Campbell KB (1994) Local Atp
Regenera-tion Is Important for Sarcoplasmic-Reticulum Ca2+
Pump Function Am J Physiol 267, C357–C366
20 Wu H, Kanatous SB, Thurmond FA, Gallardo T,
Iso-tani E, Bassel-Duby R & Williams RS (2002)
Regula-tion of mitochondrial biogenesis in skeletal muscle by
CaMK Science 296, 349–352
21 Laterveer FD, Gellerich FN & Nicolay K (1995)
Macromolecules increase the channeling of ADP from
externally associated hexokinase to the matrix of
mito-chondria Eur J Biochem 232, 569–577
22 Saks VA, Khuchua ZA, Vasilyeva EV, Belikova O &
Kuznetsov AV (1994) Metabolic compartmentation and
substrate channelling in muscle cells Role of coupled
creatine kinases in in vivo regulation of cellular
respira-tion – a synthesis Mol Cell Biochem 133–134, 155–192
23 Matsuno-Yagi A & Hatefi Y (1987) Studies on the
mechanism of oxidative phosphorylation Flow-force
relationships in mitochondrial energy-linked reactions
J Biol Chem 262, 14158–14163
24 de Groof AJ, Smeets B, Groot Koerkamp MJ, Mul
AN, Janssen EE, Tabak HF & Wieringa B (2001)
Changes in mRNA expression profile underlie
pheno-typic adaptations in creatine kinase-deficient muscles
FEBS Lett 506, 73–78
25 Anflous K, Armstrong DD & Craigen WJ (2001)
Altered mitochondrial sensitivity for ADP and
mainte-nance of creatine-stimulated respiration in oxidative
striated muscles from VDAC1-deficient mice J Biol
Chem 276, 1954–1960
26 Saupe KW, Spindler M, Tian R & Ingwall JS (1998)
Impaired cardiac energetics in mice lacking
muscle-specific isoenzymes of creatine kinase Circ Res 82,
898–907
27 Jeneson JA, Westerhoff HV & Kushmerick MJ (2000)
A metabolic control analysis of kinetic controls in ATP
free energy metabolism in contracting skeletal muscle
Am J Physiol Cell Physiol 279, 813–832
28 Jeneson JA, Westerhoff HV, Brown TR, van Echteld
CJ & Berger R (1995) Quasi-linear relationship between
Gibbs free energy of ATP hydrolysis and power output
in human forearm muscle Am J Physiol 268, 1474–
1484
29 Jeneson JA, Wiseman RW, Westerhoff HV &
Kushmer-ick MJ (1996) The signal transduction function for
oxi-dative phosphorylation is at least second order in ADP
J Biol Chem 271, 27995–27998
30 Kushmerick MJ, Meyer RA & Brown TR (1992) Regu-lation of oxygen consumption in fast- and slow-twitch muscle Am J Physiol 263, 598–606
31 Bygrave FL & Lehninger AL (1967) The affinity of mitochondrial oxidative phosphorylation mechanisms for phosphate and adenosine diphosphate Proc Natl Acad Sci USA 57, 1409–1415
32 Gellerich FN, Kapischke M, Kunz W, Neumann W, Kuznetsov A, Brdiczka D & Nicolay K (1994) The influence of the cytosolic oncotic pressure on the perme-ability of the mitochondrial outer membrane for ADP: implications for the kinetic properties of mitochondrial creatine kinase and for ADP channelling into the inter-membrane space Mol Cell Biochem 133–134, 85–104
33 Katz LA, Swain JA, Portman MA & Balaban RS (1989) Relation between phosphate metabolites and oxygen consumption of heart in vivo Am J Physiol 256, 265–274
34 Rasmussen UF & Rasmussen HN (2000) Human skele-tal muscle mitochondrial capacity Acta Physiol Scand
168, 473–480
35 Dahlstedt AJ, Katz A, Wieringa B & Westerblad H (2000) Is creatine kinase responsible for fatigue? Studies
of isolated skeletal muscle deficient in creatine kinase FASEB J 14, 982–990
36 Lawson JW & Veech RL (1979) Effects of pH and free Mg2+ on the Keq of the creatine kinase reaction and other phosphate hydrolyses and phosphate transfer reactions J Biol Chem 254, 6528–6537
37 Kemp GJ, Taylor DJ & Radda GK (1993) Control of phosphocreatine resynthesis during recovery from exer-cise in human skeletal muscle NMR Biomed 6, 66–72
38 Roef MJ, Reijngoud DJ, Jeneson JA, Berger R &
de Meer K (2002) Resting oxygen consumption and
in vivo ADP are increased in myopathy due to complex
I deficiency Neurology 58, 1088–1093
39 Lindstedt SL, McGlothlin T, Percy E & Pifer J (1998) Task-specific design of skeletal muscle: balancing muscle structural composition Comp Biochem Physiol B Bio-chem Mol Biol 120, 35–40
40 Cairns CB, Walther J, Harken AH & Banerjee A (1998) Mitochondrial oxidative phosphorylation thermody-namic efficiencies reflect physiological organ roles Am J Physiol 274, 1376–1383
41 Sims NR (1990) Rapid isolation of metabolically active mitochondria from rat brain and subregions using Per-coll density gradient centrifugation J Neurochem 55, 698–707
42 Reynafarje B, Costa LE & Lehninger AL (1985) O2 solubility in aqueous media determined by a kinetic method Anal Biochem 145, 406–418
43 Korzeniewski B & Mazat JP (1996) Theoretical studies
on the control of oxidative phosphorylation in muscle mitochondria: application to mitochondrial deficiencies Biochem J 319, 143–148
Trang 1044 Gellerich FN, Laterveer FD, Korzeniewski B, Zierz S &
Nicolay K (1998) Dextran strongly increases the
Michaelis constants of oxidative phosphorylation and of
mitochondrial creatine kinase in heart mitochondria
Eur J Biochem 254, 172–180
45 Gerhardt W (1974) Methods of Enzymatic Analysis
(Bergmeyer HU, ed), pp 510–518 Wiley-VCH,
Wein-heim
46 Vassault A (1974) Methods of Enzymatic Analysis
(Berg-meyer HU, ed), pp 119–126 Wiley-VCH, Weinheim
47 Shepherd D & Garland PB (1969) The kinetic properties
of citrate synthase from rat liver mitochondria Biochem
J 114, 597–610
48 Shephard EH & Hubscher G (1969) Phosphatidate biosynthesis in mitochondrial subfractions of rat liver Biochem J 113, 429–440
49 Schmidt B, Wachter E, Sebald W & Neupert W (1984) Processing peptidase of Neurospora mitochondria Two-step cleavage of imported ATPase subunit 9 Eur J Bio-chem 144, 581–588