From the Department of Cell Biology, Nijmegen Center
for Molecular Life Sciences, University Medical Center, University
of Nijmegen, The Netherlands and the § Division of
Cardiovascular Diseases, Departments of Medicine, Molecular
Pharmacology, and Experimental Therapeutics, Mayo Clinic, Rochester,
Minnesota 55905
Received for publication, November 11, 2002, and in revised form, January 14, 2003
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ABSTRACT |
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Genetic ablation of adenylate kinase 1 (AK1), a
member of the AK family of phosphotransfer enzymes, disturbs muscle
energetic economy and decreases tolerance to metabolic stress,
despite rearrangements in alternative high energy phosphoryl transfer
pathways. To define the mechanisms of this adaptive response, soleus
and gastrocnemius muscles from AK1 The metabolic status of skeletal muscle is reciprocally linked to
fiber-type composition and functional demand. This implies that
myocytes must undergo constant reprogramming of their gene expression
in response to fluctuations in intrinsic or extrinsic physiological
signals such as intracellular Ca2+ concentrations, hormonal
stimulation, or altered workload (1-3). Moreover, disturbance of
cellular energetics by metabolic inhibitors or genetic mutation may
also induce alterations in the muscle phenotype via changes in the gene
program for fiber-type specification (4-6). The high plasticity in the
responsiveness of muscle is exemplified by myosin isoform transitions,
mitochondrial division, and alterations in gene expression of enzymes
involved in oxidative and glycolytic pathways (3, 7-10). Although many
other aspects of myocyte infrastructure may change in concert, the
molecular mechanisms regulating reprogramming of muscle energetics are
still unknown.
Indeed, an important unresolved issue is the relationship between
muscle design and metabolic pathways maintaining cellular energy
homeostasis. Adenylate kinases
(AK,1 EC 2.7.4.3) are
evolutionary strongly conserved enzymes that catalyze the reaction ATP + AMP Along with the AK circuit, the creatine kinase (CK, EC
2.7.3.2)/phosphocreatine (PCr), nucleoside diphosphokinase (NDPK or
nm23, EC 2.7.4.6), and glycolytic phosphotransfer systems coexist in
skeletal muscle and serve to balance adenylates at ATP-consuming
and ATP-generating intracellular sites (4, 13, 15-17). The relative
importance of the AK, CK, and glycolytic phosphotransfer system is
muscle fiber-type-dependent (3, 18, 19). For example, the
CK/PCr phosphotransfer circuit is most active in fast-twitch fibers as
demonstrated by a high content of PCr and high levels of cytosolic
muscle-type (M)-CK. Slow-twitch fibers exhibit lower amounts of PCr and
M-CK, but because of the high mitochondrial content possess relatively
large amounts of ScCKmit (20). Expression of glycolytic proteins is
most abundant in fast fibers and the mechanisms by which glycolytic
genes are collectively or individually activated have in part been
identified (21). In fact, the redox cofactors NAD(P)H and
NAD(P)+, which couple glycolysis or the pentose phosphate
cycle to the Krebs cycle and oxidative phosphorylation in mitochondria,
are important regulators of glycolytic gene transcription (22). Other
coupling exists with calcium calcineurin (1),
calcium/calmodulin-dependent protein kinase (9, 10), and
AMP-activated kinase (AMPK) signaling (6), which regulate myocyte
programs for mitochondrial biogenesis and glycolytic machinery. The
mechanism underlying the regulation and distribution of AK and NDPK
gene products in specific fiber-types of muscle is less clear.
We have recently demonstrated that inactivation of the AK circuit
induces flux redistribution in the cellular phosphotransfer network,
associated with an elevated glycolytic metabolism (23). Preserved
muscle function, albeit with lower efficiency, in these animals
suggests metabolic and cellular adaptations induced by genetic stress
associated with AK deficiency. Indeed, we here uncovered a coherent
reprogramming in the genetic and molecular profile of soleus and
gastrocnemius muscles from mice lacking AK1. These adaptations occur at
the mRNA and/or protein level and could support energy metabolism
and performance in muscles compromised by AK1 deficiency.
AK1 Knockout Mice--
Gene-targeted mice carrying a
HygroBR replacement mutation in the exon-3-5 region of the
AK1 gene were derived as described in detail elsewhere (23).
Age and sex matched (3-5 months old; born on identical dates)
homozygous AK1-deficient and wild-type control animals (both with
50-50% C57BL/6 × 129/Ola mixed inbred background) were used
throughout experiments. Housing conditions were kept exactly identical
to exclude effects of variations in (steroid) hormone levels, or food
supply. Growth rates of wild-type and AK1 knockout mice were similar,
resulting in body weights varying between 26-28 and 22-24 g for
3-5-month-old males and females, respectively. The investigation
conformed to the Guidelines for the Care and Use of Laboratory Animals
of the Dutch Council and was approved by the Institutional Animal Care
and Use Committee at the University of Nijmegen.
Macroarray Hybridization--
Total RNA from freshly isolated
skeletal gastrocnemius and soleus muscles was extracted using the
lithium chloride-urea method (23). Macroarrays were prepared by
spotting individual plasmids with cDNA insert (134 different mouse
sequences for glycolytic enzymes, mitochondrial enzymes and
transporters, fatty acid metabolism enzymes, muscle regulatory factors,
proteins active in Ca2+ signaling, glucose transport,
tissue oxygenation as well as cytoskeletal components and
transcription factors (see www.ncmls.kun.nl/celbio/data.htm for
detailed information) onto Hybond N+ membranes using a
gridding robot (24). 32P-labeled single-stranded cDNA
was used as probe to hybridize membranes with gridded cDNA arrays as
previously described in detail elsewhere (24). Four AK1 knockout and
control mice were analyzed and statistically compared.
Data Analysis--
Acquisition of radioactive signals from the
gridded arrays was performed on a Bio-Rad GS 363 phosphorimager using
the Molecular Analyst software from Bio-Rad. Because the signal
intensity varied widely, membranes were exposed for 30 min (detection
of abundant RNA) and 16 h (non-abundant RNA) and hybridization
levels for individual cDNAs expressed as the mean Arbitrary Units
(A.U.) of duplicate spots. Signals were divided by the value for the sum of detectable signals, resulting in the identification of an
unaffected subset of genes that were used for final normalization.
Western Blot Analysis--
Gastrocnemius and soleus muscles
excised from the hind legs of 3 mice were pooled. Protein extracts were
prepared and analyzed as described (25). For Western blot analysis,
extracted proteins were separated on 10% SDS-polyacrylamide gels and
proteins electrophoretically transferred onto nitrocellulose membranes.
Specific proteins were detected using antibodies raised against
species-specific protein or synthetic peptides of corresponding
proteins as described elsewhere (25). After image acquisition with a
densitometer (Bio-Rad GS-690), proteins were quantified using the
Molecular Analyst software from Bio-Rad. For correlation, intensities
of signals were compared with intensities of signals in the C57BL/6
calibration series. To confirm equal loading of protein a
representative gel was stained with Coomassie Brilliant Blue (R-250)
and, after immunodetection, blots were stained with Ponceau S.
Northern Blot Analysis--
RNA blots were prepared as described
(23) and hybridized with 32P-labeled cDNAs for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), muscle pyruvate
kinase (PK-M), Enzyme Activity--
Protein extracts were prepared from frozen
powdered muscles as described in detail elsewhere (26). AK activity in
supernatant, expressed as relative light units (RLU)/min/mg protein,
was measured by a luciferase-based ATP bioluminescence assay (Sigma) in
the presence of 2 mM ADP using a BioOrbit 1253 luminometer.
NDPK activity, expressed as RLU/min/mg protein, was determined with the
same bioluminescence assay in the presence of the AK inhibitor,
diadenosine pentaphosphate (Ap5A; 200 µM),
and the reaction started with dGTP (2 mM). Three animals
per genotype were analyzed and statistically compared. The activity of
GAPDH, PK, and 3-phosphoglycerate kinase (PGK) were measured using
coupled enzyme assays (27). Reactions were started by the addition of
10 µl (GAPDH and PGK) or 20 µl (PK) of muscle extract and
activities spectrophotometrically recorded at 340 nm with a Beckman DU
7400 spectrophotometer. Per genotype, five animals were analyzed and
statistically compared.
Electron Microscopy and Morphometric Analysis--
Mice were
anesthetized with 2,2,2,-tribromorthanol (350 mg/kg intraperitoneal)
and the GPS muscle complex was fixed using a clamp and dissected during
immersion fixation with 2% glutaraldehyde in 0.1 M
phosphate buffer, pH 7.4. Subsequently, muscles were prepared and
examined on a JEOL JEM1010 electron microscope (4, 15).
Intermyofibrillar mitochondrial volumes in superficial gastrocnemius
muscle were estimated from electron micrographs at a magnification of
×8000 by point counting. For each individual muscle, at least three
randomly taken micrographs were analyzed. Per genotype, six animals
were measured and statistically compared.
Statistics--
Data are presented as mean ± S.E.
Student's t test for unpaired samples was used for
statistical analysis, and p < 0.05 was considered significant.
mRNA Profiles of Fast-twitch and Slow-twitch Fibers--
Prior
to typing the cellular and molecular adaptations that are evoked as a
response to AK1 deficiency we established the normal gene expression
characteristics of two distinctly different skeletal muscle types,
gastrocnemius and soleus muscles. These muscles can be considered the
archetypes of fast and slow skeletal muscle in the mouse hind leg (19),
for which differences in contractile performance and metabolic design
are mirrored in their transcription profiles (1, 3). Using a customized
cDNA macroarray assay and experimental conditions that avoid
mRNA amplification (24) a selective subset of mouse-muscle
cDNAs was probed for their potential to discriminate between
gastrocnemius and soleus. Ultimately, 86 candidate cDNAs were
chosen that belonged to different functional clusters, gave good signal
to noise ratio, and were considered suitable indicators for inter- and
intramuscular comparison. About 65% of the signals (56 signals)
represented mRNAs with similar expression levels in soleus and
gastrocnemius muscles. The remaining signals (30 signals) represented
mRNAs with expression levels that were significantly different
(p < 0.05; n = 4). Transcripts with
expression levels that differed by a magnitude of 2 or more (p < 0.01; n = 4) between these
two muscles are shown in Table I and Fig.
1. Quantitative data for the remaining 10 mRNAs, which differed by a magnitude less than 2 can be found at
www.ncmls.kun.nl/celbio/data.htm.
Differential regulation of energy generation, consumption and
distribution pathways is central to the determination of the nature and
function of the muscle fiber (1-3). We therefore assessed the
expression level of mRNAs for several key enzymes in these pathways. The relative abundance of AK1 mRNA was 2.2-fold higher in
the gastrocnemius muscle compared with the soleus muscle (Fig. 2A). This result was confirmed
by the observation that AK-catalyzed phosphotransfer activity was
~3-fold higher in gastrocnemius muscle than in soleus muscle (Fig.
2A). Also the composition and function of the CK
phosphotransfer system depends on the specific muscle type (20). Our
macroarray analysis showed that M-CK mRNA is 3.7-fold higher
expressed in gastrocnemius compared with the soleus muscle. In
contrast, the ScCKmit gene expression level is 2.1-fold higher in
soleus muscle (Fig. 2B). Still, the ScCKmit mRNA level was only 3 and 23% of that of M-CK mRNA in gastrocnemius and
soleus muscle, respectively. Subsequent comparison of M-CK and ScCKmit protein levels in soleus and gastrocnemius muscle by means of quantitative blot analysis, using specific antibodies directed against
ScCKmit and CK-MM (Fig. 2B, inset), indicated
that mRNA and protein product levels were well correlated. When
normalized to total protein content, the M-CK protein level appeared
1.8-fold higher in gastrocnemius than in soleus, while the ScCKmit
protein level was 2.7-fold higher in soleus than in gastrocnemius
muscle.
Along with the AK- and CK-catalyzed high-energy phosphoryl transfer,
the NDPK phosphotransfer pathway warrants transport and distribution of
~P over nucleotide di- and triphosphates (13, 16, 17). The expression
level of different NDPK isoforms present on the gridded membranes
(nm23-M1, nm23-M2, nm23-M4 and the human homologue of mouse nm23-M3,
DR-nm23), did not differ more than 1.2-fold between gastrocnemius and
soleus muscles. Taken into account that the expression of the nm23-M2
isoform was the highest in skeletal muscle (data not shown), this
finding was corroborated by the observation that there was a good
correlation between the transcript level for nm23-M2, and the
accompanying total NDPK activity (Fig. 2C). The total NDPK
activity was not significantly different between soleus and
gastrocnemius. Thus, whereas NDPK gene product and activity levels are
equally maintained in soleus and gastrocnemius muscle, AK- and
CK-mediated phosphoryl transfer activity is clearly dependent on muscle phenotype.
The sarcomeric myosin molecule is a hexamer consisting of two myosin
heavy chains (MHCs), two essential myosin light chains (ELCs), and two
regulatory myosin light chains (RLCs). Because myofibrillar protein
isoforms generally show tissue-specific distribution these proteins may
serve as useful markers for skeletal muscle fiber typing. As
anticipated on the basis of a slow or fast-twitch muscle fiber (3), the
expression level of genes encoding the MLC-1s/v and MLC-2s mRNAs
differed 6-8-fold between gastrocnemius and soleus muscle (Table I and
Fig. 1). Also the signals for the mRNAs for mitochondrial inorganic
phosphate carrier (PiC), Ca2+-ATPase isoform Serca2, and
myoglobin, of which the translation products are associated with
oxidative metabolism, were 2.0-, 3.8-, and 5.2-fold higher in the
soleus muscle, respectively (Table I and Fig. 1). Strikingly, the
steady-state transcript level for the nuclear-encoded mitochondrial
transcription factor Tfam (or mtTFA) was 3.9-fold higher in the
glycolytic gastrocnemius muscle compared with the oxidative soleus
muscle. This observation was surprising because the gastrocnemius is a
mitochondria-poor muscle, but may be explained by the fact that
cooperation of more than one factor is needed to regulate mitochondrial
biogenesis (9, 10). Unexpectedly, relatively high steady-state B-CK transcript levels were detected in soleus and gastrocnemius muscle. This observation is most easily explained by the fact that immature muscle cells (i.e. satellite cells) actively transcribe the
B-CK gene (15). As expected, signal intensities for
mRNAs related to glycolysis ( Differences in Gene Products for High Energy Phosphoryl Transfer in
the Absence and Presence of AK1--
Changes at the molecular level
may occur when the muscle genotype or the integrity of various
intrinsic or extrinsic physiological control mechanisms are disturbed
(6, 9, 25, 28). To determine the spectrum of molecular changes related
to the absence of AK1 we compared mRNA profiles of gastrocnemius
and soleus, between wild-type and AK1
Knockout of AK1 produced a dramatic 96% decrease in the AK1 mRNA
signal (Table II and Fig. 3, A-B) for gastrocnemius muscle. Likewise, soleus muscle of AK1 Adaptation in Glycolytic, Mitochondrial, and Structural Gene
Product Levels--
Several studies support the functional interaction
of the AK circuit with the glycolytic machinery for energy production
(13, 29-32). Previous studies showed that AK1
In addition to glycolytic mRNAs also transcripts encoding enzymes
in other related catabolic pathways were analyzed. In the glycolytic
gastrocnemius muscle of AK1 knockouts we observed lower mRNA levels
for long chain acyl-CoA dehydrogenase and glutamate dehydrogenase (35 and 54% reduction, respectively; Table II and Fig. 3B).
Both messengers encode proteins that reside in the mitochondrial matrix
and are involved in the oxidation of glutamate and fatty acids. In the
oxidative soleus muscle these messenger signals were unaffected by the
absence of AK1 (data not shown).
Based on the intramuscular comparison (Table I and Fig. 1) the
regulatory cardiac/slow-twitch myosin light chain 2 (MLC-2s) may be
considered a marker for oxidative fiber-types. In AK1 animals, signals
representing the MLC-2s mRNA (full-length and partial) were
downregulated by ~40% in the gastrocnemius muscle, but not affected
in soleus muscles (Table II and Fig. 3B). This observation suggests that down-regulation of MLC-2s gene transcription
is essential in evoking changes in isoenzyme composition for the regulatory MLCs. Interestingly, also the essential cardiac/slow-twitch MLC-1s/v transcript in gastrocnemius muscle inclined to a decrease in
concentration (50% reduction; p < 0.07;
n = 4).
Among the key factors involved in transcriptional control of myofibers
are the myogenic basic helix-loop-helix (bHLH) transcription factors.
Expression levels of MyoD and Myf5 strongly correlate with the fast
muscle phenotype (35, 36), while myogenin has been shown to be
associated with the slow muscle phenotype (37). In fast-twitch
gastrocnemius muscles lacking AK1 the MYF5 transcript level was
increased by more than 200% (Table II and Fig. 3B). Whereas
signals of myogenin transcripts could not be reliably detected, the
MyoD mRNA level tended to be up-regulated (73% with low
significance p < 0.09; n = 4). In the
soleus muscle no differences were observed for the bHLH transcription factors.
Northern blot analysis of newly isolated mRNA samples from
gastrocnemius muscles independently confirmed the results from our
macroarray experiments (Fig. 3D). Signals obtained with
probes specific for ScCKmit and GLUT4 were at similar strength for
wild-type and AK1-deficient muscles (0.68 ± 0.05 versus 0.68 ± 0.04 and 0.37 ± 0.02 versus 0.36 ± 0.02 A.U. in AK1KO and wild type,
respectively; p > 0.05; n = 4). The
intensities of mRNA bands for Increased Glycolytic Phosphotransfer Capacity in AK1 Knockout
Skeletal Muscle--
We next used protein-chemical and functional
assays to establish the relationship between mRNA and protein
levels and enzymatic activities, focusing on key phosphoryl transfer
enzymes in the glycolytic pathway, PK, GAPDH, and PGK. Semiquantitative
blot and enzymatic analysis of GAPDH showed maintained protein levels and activities for GAPDH in mutant skeletal muscle (Fig.
4A). The protein level for PK
was increased 2-fold in the absence of AK1 and was paralleled by 2-fold
increase in PK-catalyzed phosphotransfer capacity (Fig. 4,
B-D). Thus, the increase in GAPDH mRNA level is not
followed by an increase in GAPDH protein quantity and enzymatic activity. For the adaptive PK up-regulation in AK1
In addition to PK, PGK is the other principal phosphoryl transfer
enzyme that produces ATP in the glycolytic pathway. PGK catalyzed
phosphotransfer activity was increased 2-fold (Fig. 4E).
Together, the increased steady-state levels for several glycolytic mRNA species and the more direct observation of increased enzymatic activity of PK and PGK, indicate that glycolysis-driven phosphoryl exchange between ADP and ATP may be one of the principal targets for
adaptation in fast-twitch muscles of AK1 AK1-deficient Muscle Show Increased Mitochondrial
Content--
Based on theoretical considerations and experimental
evidence it is now commonly accepted that the AK, CK, and glycolytic circuits may be intertwined with mitochondrial activity and together form integrated networks for high energy phosphoryl transfer at different subcellular locales (13, 16, 23, 32). As shown in Fig.
5A and inset
4C, the level of the cytosolic M-CK protein was not affected
in skeletal muscle. We did, however, observe a 40% increase in the
level of mitochondrial ScCKmit in the gastrocnemius muscle of
AK1-deficient mice. Also, the signal for the mitochondrial adenine
nucleotide transporter (ANT) was similarly increased. Similar results
were obtained for another fast-twitch muscle, the psoas major muscle
(data not shown). Intriguingly, the increase in steady-state protein
levels of ANT and ScCKmit in AK1 knockout gastrocnemius muscle was not
accompanied by a corresponding increase in mRNA level. The level of
two other mitochondrial proteins, the inorganic phosphate carrier (PiC)
and voltage-dependent anion channel (VDAC; porin), were not
changed in AK1-deficient gastrocnemius muscle (data not shown). In
mutant soleus muscle no significant alterations in the level of ANT and
ScCKmit protein were detected.
To analyze whether the changes in mitochondrial marker proteins were
also reflected in variation of mitochondrial density or appearance we
applied EM-morphometric analysis. Indeed, a 2-fold increase in
mitochondrial volume was detected in AK1-deficient gastrocnemius
muscles (Fig. 5, B and C). Taken together, these findings indicate that in response to AK1 gene deletion cell
type-dependent adaptations occur to maintain cellular
energetic homeostasis.
Previously we had demonstrated that inactivation of the
AK1 gene compromises economic efficiency of the cellular
energetic network despite the fact that there is rewiring of fluxes
through other pathways for metabolic energy transfer. These adaptive
responses apparently ameliorate, but do not entirely obscure, the
effects of loss of the AK1 phosphotransfer activity (23, 26, 38). Here
we provide a detailed analysis of the molecular events underlying compensatory responses to muscle AK1 deficiency, demonstrating that
regulation at the level of gene transcript abundance, enzymatic activity, as well as (re)organization of the cellular ultrastructure is
involved, with a signature to sustain cellular high energy phosphoryl
generation and transfer capacity.
Although regulation of the AK1 gene and its products has
been described in several reports (23, 39), not much attention has been
paid to the relevance for metabolic context or cell type requirements
of AK-mediated phosphoryl exchange. We demonstrate here by mRNA
profiling and biochemical activity measurement that AK1 transcript
level is well correlated to enzymatic capacity (when normalized to
total RNA or protein content) and is 2-3-fold higher in fast-twitch
gastrocnemius compared with slow-twitch soleus muscle. This underscores
the relative importance of AK catalysis for muscle that is relatively
poor in mitochondria and highly dependent on glycolytic ATP production.
Similar fiber-type specificity was noted for mRNA and protein
products of the gene for muscle-type cytosolic creatine kinase, M-CK.
Although we still do not fully understand how cytosolic AK and CK
enzymes are integrated in the cellular energy network (13, 32),
predominance in fast-twitch myocytes would fit to their role in
protecting the cell from threshold effects of abnormal ATP/ADP/AMP
ratios during transient periods of sudden and profound energy demand.
No correlation to fiber-type was found for expression of members of the
NDPK family of genes (nm23-M1, nm23-M2, nm23-M3/DR-nm23, and nm23-M4).
Based on this finding it is tempting to speculate that NDPK-mediated
phosphotransfer may feed high-energy phosphoryls into other metabolic
pathways, not directly involved in energy homeostasis associated with
muscle contraction.
Our array profiling demonstrated that AK1 absence caused a parallel,
1.5-2-fold, increase in the level of PK-M, Various regulatory circuits like the myogenic bHLH transcription
factor family, calcineurin/CaMK/PGC-1/NFAT/MEF2, myogenic regulatory
factor (MRF) activity, or NPAS2/BMAL pathways have now been identified
that, together, may be implemented in the transcriptional regulation of
cellular carbohydrate metabolism and ultrastructural design of
myofibers (and other cell types as well) (1, 9, 10, 22, 35-37, 40). We
found no change in abundance of mRNA for calcineurin in
AK1 It is of note that different pathways for matching muscle
infrastructure to conditions of metabolic stress might be effective in
slow and fast type myofibers. In the highly oxidative soleus muscle,
lack of AK1 produced an 80% increase in the steady-state level of
In validating our array quantification data with Northern and Western
blot analyses we noticed that there was discordance between mRNA
and protein levels for some genes. Most striking was that the almost
2-fold increase in GAPDH mRNA was not paralleled in GAPDH activity
in muscle extract. Conversely, a relatively modest increase in PK-M
mRNA level was accompanied by a 2-fold increase in PK-M content and
activity. This suggests that the mechanism(s) of adaptive response may
also include regulation at the rate of translation or protein turnover
(44). Ultimately, shifts in isoenzyme composition, phosphorylation or
protein complexation should therefore be taken into account. For
example, skeletal muscle PK-catalyzed phosphotransfer activity can be
increased upon binding with MM-CK resulting in an increased flux
through PK, independently of its substrate concentrations (45).
Interestingly, an up-regulation of GAPDH and/or PK mRNA, as we
found here, has also been reported for heart and skeletal muscle in
response to muscle disuse and ischemic stress (46, 47).
The adaptations in the glycolytic pathway, especially the
phosphotransfer enzymes PK and PGK, should also be discussed in the
context of a possible direct structural and functional association between AK1 and glycolytic enzymes. Indeed, AK1 can physically interact
with phosphofructokinase and participate in the formation of a larger
glycolytic enzyme complex or cluster (30, 31, 48). Whether this complex
only serves to provide localized glycolytic ADP:ATP phosphotransfer
capacity, presumably important for sustaining actomyosin sliding and
force production, or also has any other structural-organizational
function is currently unknown. Immunostaining experiments have
indicated that AK1 is present in distinct subcellular locales of
skeletal muscle that coincide with localization of enzymes of the
glycolytic apparatus (49). Adaptations, instead of serving a general
role in cellular energetics, may therefore also have a role in guarding
structural integrity of the glycolytic infrastructure of muscle.
Finally, it is important to note that AK1 is involved in the
communication between myofibrillar ATPases and mitochondria (13, 27,
32, 50), thereby maintaining efficient intracellular energy flow (23).
Translocation of ADP into mitochondria or release of ATP in the cytosol
is achieved via translocator complexes composed of ANT, porin (VDAC)
and octameric mitochondrial creatine kinase (51). Because diffusion of
ADP is very limited in muscle cells (52) and the AK pathway for ADP
(re)phosphorylation is depleted in AK1/
mice were
characterized by cDNA array profiling, Western blot and
ultrastructural analysis. We demonstrate that AK1 deficiency induces
fiber-type specific variation in groups of transcripts involved in
glycolysis and mitochondrial metabolism and in gene products defining
structural and myogenic events. This was associated with increased
phosphotransfer capacities of the glycolytic enzymes pyruvate kinase
and 3-phosphoglycerate kinase. Moreover, in AK1
/
mice,
fast-twitch gastrocnemius, but not slow-twitch soleus, had an increase
in adenine nucleotide translocator (ANT) and mitochondrial creatine
kinase protein, along with a doubling of the intermyofibrillar mitochondrial volume. These results provide molecular evidence for wide-scale remodeling in AK1-deficient muscles aimed at
preservation of efficient energetic communication between ATP producing
and utilizing cellular sites.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
2 ADP. This reaction is one of the principal steps in adenine
nucleotide metabolism and high energy phosphoryl (~P) transfer in the
cellular bioenergetic network (11-13). Among several AK isoenzymes
found in mammals, skeletal muscle is particularly rich in AK1, the
major cytosolic isoform (14).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-enolase, MLC-2s, ScCKmit, GLUT4, and
-actin as
probe (probe length, 0.5-2 kbp). Hybridization was carried out
overnight at 68 °C in 0.5 M NaPO4 buffer, pH
7.2, 7% (w/v) SDS, 1 mM EDTA, and blots were washed to a
final stringency of 0.1× SSC/1% (w/v) SDS at 68 °C and exposed to
Kodak X-Omat films. Signals were quantified by phosphorimager analysis
as described above. Per genotype, four animals were analyzed
(independently from macroarray experiments) and statistically compared.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Differential mRNA expression in gastrocnemius and soleus
skeletal muscle
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Fig. 1.
Fiber-type specific mRNA expression.
A, scatter graph representation of array data from
gastrocnemius (x-axis) compared with soleus
(y-axis). Each data point represents the mean of signals
obtained for an individual gene. Transcript levels are represented by
the (x,y) positions in the scatter graph. Data points marked
with gray boxes represent differences by a magnitude of 2 or
more. The solid line indicates maintained steady-state
mRNA levels whereas the dotted lines indicate mRNA
levels that differ by 2-fold. B, net differences in mRNA
expression level between the gastrocnemius and soleus muscle. Note the
relative higher mRNA expression of AK1 in the gastrocnemius muscle
(no. 9). Data are expressed as the mean ± S.E. from four determinations.
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Fig. 2.
Fiber-type distribution of AK, CK, and
NDPK. A, relative abundance of AK1 transcript and total
AK enzymatic activity in soleus and gastrocnemius muscles.
B, relative abundance of ScCKmit and M-CK in soleus and
gastrocnemius muscles. The inset shows a Western blot of
immunoreactive ScCKmit and M-CK from soleus (S) and
gastrocnemius (G) muscles. C, relative abundance
of NDPK-M2 transcript and total NDPK enzymatic activity in soleus and
gastrocnemius muscles. Data are expressed as the mean ± S.E. from
three or four determinations.
-enolase, aldolase A, GAPDH, and
PGK) or coupled pathways (glycogen phosphorylase B, LDH-A, and GPDH)
were higher in gastrocnemius than in soleus muscle (Table I and Fig.
1). Thus, we may conclude that the cDNA macroarray has sufficient discriminating power to distinguish mRNA profiles of fast and slow
type muscles.
/
animals.
Altogether, fourteen genes were identified for which the expression
levels differed significantly between AK1-proficient and
-deficient muscles (Table II and Fig.
3).
Transcript profiling in wild-type and AK1/
gastrocnemius
muscle
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Fig. 3.
Expression profiling of skeletal muscles from
AK1 /
and wild-type mice. A,
autoradiograms of cDNA array profiles from wild-type and AK1
knockout gastrocnemius muscle. Total RNA was isolated, and first strand
cDNA product was 32P-labeled and used as hybridization
probe. Signal intensities of individual spots were quantified by
phosphorimager analysis and normalized to the sum of detectable
signals. Note the absence of AK1 mRNA in AK1 knockout muscle
(position B15). B, scatter graph for the comparison of
transcript profiles between AK1 mutant and wild-type gastrocnemius
muscles. Each data point represents the mean ± S.E. of signals
obtained for an individual gene from four determinations. Transcript
levels are represented by the (x,y) positions in the scatter
graph. X-axis and y-axis represent wild-type and AK1
knockout RNA profiles, respectively. The solid line
indicates maintained steady-state mRNA levels. Significant
different mRNA levels are marked with gray boxes.
C, scatter graph for comparison of transcript profiles
between AK1 mutant and wild-type soleus muscles. D, Northern
blots of total RNAs isolated from wild type (n = 4) and
AK1 knockout (n = 4) gastrocnemius muscles. Specific
probes for ScCKmit, GLUT4, GAPDH, PK-M,
-enolase, and
-actin were
used for hybridization. RNA levels were quantified by phosphorimager
analysis and normalized to the
-actin signal (see text).
/
animals showed a 95%
reduction in AK1 mRNA content (Table II and Fig. 3C). We
surmised that transcript profiles of other enzymes involved in energy
transfer pathways might be among the principal targets for adaptation
to AK1 deficiency. Yet, M-CK transcript levels on the arrays of AK1
knockout gastrocnemius and soleus were not significantly different from
wild type. The steady-state M-CK transcript level was respectively
88 ± 8 A.U. and 85 ± 12 A.U. in the wild type and AK1
knockout gastrocnemius muscle (n = 4, p > 0.05) and 16 ± 1 A.U and 16 ± 2 A.U. in the wild-type and AK1 knockout soleus muscle (n = 4, p > 0.05), respectively. Also the level of the
mRNA for the mitochondrial CK isoform, ScCKmit, was unaffected
(2.6 ± 0.1 versus 2.3 ± 0.1 A.U. in the
gastrocnemius and 3.5 ± 0.2 versus 3.5 ± 0.6 A.U. in the soleus of wild-type and AK1-deficient mice, respectively;
n = 4, p > 0.05). Thus, in response to
AK1 gene deletion, the levels of transcripts encoding the
ScCKmit and M-CK isoforms are not changed. We observed, however, a
2-fold increase in the signal level for DR-nm23 mRNA in the absence
of AK1 in gastrocnemius muscle (Table II and Fig. 3B). DR-nm23 mRNA level in soleus muscle did not differ by genotype. Total NDPK-catalyzed phosphoryl transfer capacity in the gastrocnemius and soleus muscles was not affected by AK1 gene
deletion (21 ± 2 versus 20 ± 2 .103
RLU/min/mg protein and 23 ± 2 versus 27 ± 2 .103 RLU/min/mg protein in wild-type and AK1 knockout,
respectively; p > 0.05, n = 3).
/
muscles
have up-regulated glycolytic flux. In keeping with this finding, we
observed that four of the affected signals in gastrocnemius represented
mRNAs that encode enzymes acting in the glycolytic pathway. For
muscle enolase, consisting of two subunits encoded by two distinct
genes (enolase
and
), there was a significant increase in the
steady-state level of the
-enolase mRNA in the gastrocnemius
knockout muscle (Table II and Fig. 3B). In soleus muscle a
similar increase, but now for
-enolase mRNA, was seen (Table II
and Fig. 3C). As the
-enolase level is dependent on muscle energetic demand (33, 34), this result suggests a similar role
for the
-enolase subunit in skeletal muscle. Similarly, the levels
of mRNAs for PK-M, GAPDH, and GPDH were ~1.5-fold
increased in mutant gastrocnemius compared with wild type (Table II and Fig. 3B). Also the PGK mRNA level tended to be greater
(30%) in the AK1-deficient gastrocnemius muscle (p < 0.08). Conversely, there was a 23% decrease in the level of the
mRNA for the tetrameric glycolytic enzyme lactate dehydrogenase
(LDH), LDH-A, in soleus muscle in AK1
/
compared with
wild type (Table II and Fig. 3C). Levels of mRNAs for
PK-M, GAPDH, and GPDH in soleus and LDH-A in gastrocnemius did not
significantly differ between knockout and wild type muscles. Because
the flux through glycolysis was increased in
gastrocnemius-plantaris-soleus (GPS) muscle complex of AK1
knockout mice (23) we next raised the question whether the mRNA
level for the insulin and AMPK-dependent glucose importer,
GLUT4, was possibly altered. The steady-state GLUT4 transcript level
appeared unaffected by AK1 absence in both gastrocnemius (1.8 ± 0.4 and 2.9 ± 0.7 A.U. in wild type and AK1 knockout,
respectively; p > 0.05, n = 4) and
soleus (1.4 ± 0.3 and 1.4 ± 0.4 A.U. in wild type and AK1
knockout, p > 0.05; n = 4) muscle.
-enolase and PK-M were 1.3- and
1.4-fold up-regulated in mutant gastrocnemius muscle (2.1 ± 0.1 versus 1.6 ± 0.1 and 1.01 ± 0.02 versus 0.71 ± 0.04 A.U. in AK1KO and wild type,
respectively; p < 0.03; n = 4). Also
the GAPDH mRNA signal was increased 1.4-fold in mutant gastrocnemius (0.46 ± 0.03 versus 0.34 ± 0.02 A.U. in AK1KO and wild type, respectively; p < 0.02;
n = 4), but less pronounced than in the macroarray data
set (1.8-fold increase). Use of the MLC-2s cDNA probe confirmed the
earlier observed decrease in MLC-2s transcript level (signal intensity
0.8 ± 0.2 versus 1.5 ± 0.2 A.U. in AK1KO and
wild type, respectively; p = 0.05; n = 4). Independent confirmation of the mRNA array data for soleus
muscle was not achieved because the RNA yield from this muscle was too
low for use in the Northern blot assay.
/
gastrocnemius muscle mRNA level, protein content as well as
enzymatic activity appeared linearly coupled.
View larger version (22K):
[in a new window]
Fig. 4.
Increased glycolytic phosphotransfer capacity
in AK1 /
gastrocnemius skeletal muscle.
A, GAPDH activity in wild-type and AK1 knockout
gastrocnemius muscles (n = 5 each). The
inset illustrates a Western blot image of immunoreactive
GAPDH in wild-type and AK1 knockout gastrocnemius muscle. B,
densitometric analysis of Western blot image (inset) for
PK-M. Protein extracts were pooled from three mice, electrophoretically
separated on 10% SDS-polyacrylamide gels, and subjected to Western
blot analysis. WT (
) and AK1KO (
) PK-M protein levels are
indicated on the linear calibration plot. This plot serves to correlate
signal intensities with known quantities of immunoreactive PK-M
protein. Note the 2-fold-increased PK-M protein level in AK1KO (1.6)
compared with wild type (0.8) gastrocnemius protein extracts.
C, densitometric analysis of Western blot image for M-CK
(inset). The blot shown in B was reprobed with
the M-CK antibody. Note equal M-CK protein levels in wild-type (1.2)
and AK1KO (1.2) gastrocnemius protein extracts. D, PK
activity in wild-type and AK1 knockout gastrocnemius muscles
(n = 5). E, PGK activity in wild-type
and AK1 knockout gastrocnemius muscles (n = 5). Asterisk indicates significant difference between
groups.
/
mice.
View larger version (66K):
[in a new window]
Fig. 5.
Increased mitochondrial content in
AK1-deficient skeletal muscle. A, Western blot images
for ScCKmit, ANT, and M-CK. Protein extracts were pooled from three
mice, electrophoretically separated on 10% SDS-polyacrylamide gels and
subjected to Western blot analysis. Images were analyzed as described
under "Materials and Methods." B, ultrastructural
changes in gastrocnemius skeletal muscle from AK1-deficient mice.
Electron micrographs of longitudinal sections through myofibers of the
gastrocnemius muscle. Fast-twitch fibers of AK1 deficient mice display
an increased intermyofibrillar mitochondrial volume. C,
increased mitochondrial density in AK1-deficient gastrocnemius muscle.
Mitochondrial volume density was assessed by point counting for six
AK1 /
and control animals. Asterisk indicates
significant difference between groups.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-enolase, GAPDH, PGK,
and GPDH mRNAs in gastrocnemius. Northern blot quantification supported this finding for the first three mRNAs mentioned. This strongly points to concerted transcriptional regulation of these glycolytic genes. In concordance with our earlier findings (23) we
surmise that up-regulation of mRNAs encoding glycolytic enzymes in
gastrocnemius serves a general need for larger capacity of the
glycolytic pathway in AK1
/
mutants. It is of note,
therefore, that the contents of mRNAs for long chain acyl-CoA
dehydrogenase and glutamate dehydrogenase were decreased. This could
suggest that the production of Krebs cycle intermediates from metabolic
pathways other than glycolysis may be down-regulated. Again this could
be viewed as a direct adaptational effect that fits the general drift
toward a more glycolytic profile.
/
gastrocnemius and soleus, but this does not
exclude a regulatory role for this enzyme, as the calcineurin pathway
is mainly regulated at the level of factor relocation. We did observe,
however, that the concentration of MYF5, a member of the bHLH family of
myogenic transcription factors (TFs), was increased 3-fold. Also the
level of another member, MyoD, was increased. Allen et al.
(36) reported that MYF5 and MyoD preferentially activate the skeletal
MHCIIb gene, specifying the myosin isoform that is highest
expressed in the glycolytic fast-type fiber IIB. In parallel, a role
for MyoD has been proposed in the maintenance of fast-fiber
characteristics (35). Against this background, these TFs might be
considered good candidates for being involved in the transition in
glycolytic versus oxidative phenotype and the accompanying
contractile properties of muscle design in our AK1
/
mice.
-enolase mRNA and down-regulated LDH-A mRNA levels with no
changes in PK-M, GAPDH, and GPDH mRNAs levels. Intriguingly, recent
findings indicate that the
-enolase mRNA encodes two distinct proteins,
-enolase and Myc-binding protein (MBP)-1 protein, due to
alternative usage of translation initiation sites (41).
-Enolase is
involved in glycolysis, whereas the MBP-1 protein down-regulates c-Myc
oncoprotein expression. Activation by c-Myc, in turn, can promote
LDH-A gene transcription (42, 43). Increased production of
the MBP-1 protein from the
-enolase mRNA would therefore be expected to result in down-regulation of c-Myc, and subsequent reduction in LDH-A gene activity. Although this is exactly
what was observed in our array measurements, clearly more detailed study is necessary to see whether this explains our findings. Another
hypothetical possibility would be that the redox state of NAD cofactors
and its effect on the NPAS2/BMAL1 transcription machinery (22) is
involved in the metabolic signaling and suppression of the LDH-A
transcript level. We put this possibility forward because gastrocnemius
muscles of AK1
/
mice show significant higher
NAD+ levels (20%
increase).2
/
gastrocnemius
muscle, the observed 2-fold increase in mitochondrial volume, and
concomitant increase in ANT and ScCKmit protein may serve to match the
mitochondrial ADP import capacity with the increase in glycolytic flux.
Apparently, post-translational regulation may be involved in this
phenomenon, as the mRNA levels for ANT and ScCKmit were not
accordingly adapted. Intriguingly, other mitochondrial import proteins
like the PiC and VDAC were not increased. In this regard muscles of
M-CK knockout mice have a similar pattern in molecular and
cytoarchitectural adaptations (24, 25). In these mutants, ScCKmit and
ANT1 mRNA levels were maintained whereas ScCKmit and ANT protein
levels were dramatically increased. This was also paralleled by an
increase in pyruvate kinase protein level and intermyofibrillar
mitochondrial volume (24, 25). When combined, this suggests that the
AK1- and M-CK-catalyzed phosphotransfer circuit may have functional
redundancy as the deficiency for both circuits is sensed and
counteracted in a highly similar manner.
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ACKNOWLEDGEMENTS |
---|
We thank F. Oerlemans for technical assistance and Dr. M. Groot Koerkamp and Dr. H. Tabak for providing help in use of the gridding robot facility.
![]() |
FOOTNOTES |
---|
* This work was supported by the Netherlands Organization for Scientific Research (NWO-ZonMW) (901-01-095) and the Nederlandse Kankerbestrijding/KWF (KUN 98-1808).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Cell Biology, NCMLS University Medical Center, University of Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands. Tel.: 31-24-3614329/3614287; Fax: 31-24-3615317; E-mail: b.wieringa@ncmls.kun.nl.
Published, JBC Papers in Press, January 31, 2003, DOI 10.1074/jbc.M211465200
2 E. Janssen, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: AK, adenylate kinase 1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; CK, creatine kinase; M-CK, muscle-type CK; RLU, relative light unit; A.U., arbitrary units; PK, pyruvate kinase; PGK, 3-phosphoglycerate kinase; MHC, myosin heavy chain; LDH, lactate dehydrogenase; MLC, myosin light chain; GPDH, glycerol-3-phosphate dehydrogenase; NDPK, nucleoside diphosphokinase; GDH, glutamate dehydrogenase.
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