Laboratoire de Physiologie, Groupe Physiologie et Physiopathologie de l'Exercice et du Handicap Groupement d'Intérêt Public-E2S, Faculté de Médecine, 42023 Saint-Etienne, France
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ABSTRACT |
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Myogenesis requires energy
production for the execution of a number of regulatory and biosynthesis
events. We hypothesized that mitochondrial biogenesis would be
stimulated during skeletal muscle regeneration. Tibialis anterior
muscles of male Sprague-Dawley rats were injected with 0.75%
bupivacaine and removed at 3, 5, 7, 10, 14, 21, or 35 days after
injection (n = 5-7/group). Two main periods
emerged from the histochemical analyses of muscle sections and the
expression of proliferating cell nuclear antigen, desmin, and creatine
phosphokinase: 1) activation/proliferation of satellite
cells (days 3-14) and 2) differentiation
into muscle fibers (days 5-35). The onset of
muscle differentiation was accompanied by a marked stimulation of
mitochondrial biogenesis, as indicated by a nearly fivefold increase in
citrate synthase activity and state 3 rate of respiration between
days 5 and 10. Peroxisome proliferator-activated
receptor- coactivator-1 (PGC-1) mRNA level and mitochondrial
transcription factor A (mtTFA) protein level peaked on day
10 concurrently with the state 3 rate of respiration. Therefore,
transcriptional activation by PGC-1 and mtTFA may be one of the
mechanisms regulating mitochondrial biogenesis in regenerating skeletal
muscle. Taken together, our results suggest that mitochondrial biogenesis may be an important regulatory event during muscle regeneration.
mitochondrial respiration; muscle precursor cells; myogenesis; peroxisome proliferator-activated receptor- coactivator-1; mitochondrial transcription factor A
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INTRODUCTION |
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SKELETAL MUSCLE HAS A REMARKABLE CAPACITY to regenerate after injury. Different events must occur to achieve complete regeneration of the muscle: phagocytosis of muscle debris, revascularization, activation, proliferation, and differentiation of muscle precursor cells, and reinnervation (10). Experimentally, bupivacaine is frequently used to study skeletal muscle regeneration. This myotoxic agent causes severe perturbations of calcium homeostasis (31, 47) associated with hypercontractions of muscle fibers and disruptions of plasmalemma membranes (20, 44), which lead ultimately to rapid muscle fiber necrosis (18, 20). However, muscle satellite cells are resistant to the action of bupivacaine, and are thus available for the subsequent recapitulation of the myogenic program (21).
Mitochondrial biogenesis is one of the striking responses observed in
skeletal muscle exposed to a variety of physiological conditions (see
Refs. 12 and 26 for reviews). The
synthesis of mitochondrial proteins involves the expression of genes
originating from both nuclear and mitochondrial genomes. This
dual-genomic organization is coordinated by a set of transcription
factors, including peroxisome proliferator-activated receptor-
coactivator-1 (PGC-1), nuclear respiratory factor-1 (NRF-1), and
NRF-2, among others. PGC-1 stimulates the expression of NRF-1 and NRF-2
(49), whose regulatory elements are shared by many genes
encoding mitochondrial proteins (46). PGC-1 and NRF-1 also
coactivate the expression of mitochondrial transcription factor A
(mtTFA) (49), the only known regulator of mitochondrial
replication and transcription. Thus the transcription factors involved
in mitochondrial biogenesis are key contributors to the nuclear control
of mitochondrial phenotypic alteration and energy production in
skeletal muscle.
Recapitulation of the myogenic program requires energy production for the execution of a number of regulatory and biosynthesis events. Accordingly, mitochondrial biogenesis accompanies the in vitro differentiation of myoblasts into myotubes (30, 36). In addition, muscle cell differentiation appears to depend on mitochondrial function. Indeed, respiration-deficient myoblasts devoid of mitochondrial DNA fail to differentiate (23). Similarly, inhibition of mitochondrial protein synthesis with chloramphenicol prevents the differentiation of myoblasts into myotubes (22, 28). These studies illustrate the importance of the biosynthesis of functional mitochondria during in vitro myogenesis. However, much less is known about mitochondrial biogenesis during in vivo skeletal muscle regeneration.
The aim of this study was to analyze the relationship among mitochondrial content, mitochondrial function, and skeletal muscle regeneration. For this purpose, the bupivacaine-induced muscle degeneration model was employed to examine the expression of mitochondrial proteins and transcription factors in relation to muscle cell proliferation and differentiation.
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METHODS |
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Animal care and bupivacaine injection. The animal protocol was approved by the Ministère de l'Agriculture et de la Forêt. Male Sprague-Dawley rats (278 ± 2 g; n = 44) were housed individually in a temperature-controlled room (21°C) with a 12:12-h light-dark cycle and allowed food and water ad libitum. Animals were anesthetized with an intraperitoneal injection of pentobarbital sodium (60 mg/kg body wt). Injections of bupivacaine (4 × 150 µl of 0.75% bupivacaine in 0.9% NaCl) were done into the tibialis anterior (TA) muscle with a 30-gauge needle. The injection procedure used in the present study has been shown to be highly effective in eliciting a large and complete muscle degeneration (19). The contralateral TA muscle was injected with a saline solution.
Tissue sampling. After 3, 5, 7, 10, 14, 21, or 35 days of injection (n = 5-7/group), rats were anesthetized, and the entire TA muscle was removed from each limb. A first portion of muscle was embedded in cryopreservative (Cryomount, HistoLab), quickly frozen in isopentane, and stored in liquid N2 for subsequent histochemical analyses. A second portion was frozen in liquid N2 and used for total RNA and protein extractions. A third portion was used immediately for mitochondrial isolation.
Histochemical analyses.
Transverse muscle sections (10 µm) were cut in a cryostat microtome
at 20°C and stained with hemalum-eosin-safran. The area characterized by the presence of infiltrating cells, interstitial edema, necrotic fibers, proliferating myoblasts, and generalized myophagy was measured and expressed as the percentage of the whole muscle cross-sectional area. Regeneration was assessed by counting the
number of fibers with central myonuclei divided by the total number of
fibers. Analyses were performed using a light microscope connected to a
computerized image analysis system [National Institutes of Health
(NIH) Image 1.61].
Protein extraction for immunoblotting and enzyme assays.
Total proteins were extracted from powdered muscles as previously
described (14) and stored at 80°C. Concentrations were spectrophotometrically measured at 750 nm using the Bio-Rad protein assay.
Immunoblotting.
Aliquots of protein [15 µg/lane for proliferating cell nuclear
antigen (PCNA) and desmin and 30 µg/lane for mtTFA] were mixed with
a loading buffer [62.5 mM Tris, pH 6.8, 5% glycerol (vol/vol), 1%
SDS (vol/vol), 2.5% -mercaptoethanol (vol/vol)], boiled for 3 min,
applied to a 12.5% (wt/vol) SDS polyacrylamide gel, and electrophoresed at 115 V for 4 h at 4°C. Separated proteins were electrotransferred for 1 h at 4°C onto nitrocellulose membranes. The membranes were then blocked in TBS-5% milk solution (PCNA and
desmin) or in TBS-Tween 20-5% milk solution (mtTFA) at room temperature for 1 h. The following primary antibodies were
used for immunoblotting: PCNA, 1:500 dilution (PC10 NeoMarkers);
desmin, 1:500 dilution (D33, DAKO); and mtTFA, 1:500 dilution [a
generous gift of D. A. Hood and H. Inagaki, National Industrial
Research Institute, Nagoya, Japan (17)]. Primary
antibodies were incubated overnight at 4°C. Rabbit anti-mouse IgG
[1:2,000 (vol/vol); P0161, DAKO] or goat anti-rabbit IgG [1:2,000
(vol/vol); P0448, DAKO] conjugated to horseradish peroxidase was used
for chemiluminescent detection of proteins (ECL, Amersham). The films
were scanned and quantified using NIH Image 1.61.
Enzyme activities.
Enzyme activities were fluorometrically measured (exc = 340 nm
and
em = 450 nm) according to Lowry and Passonneau
(32) with some modifications. For creatine phosphokinase
activity (CPK, EC 2.7.3.2.), 10 µl of protein extract (1:100
dilution, vol/vol) were added to 960 µl of assay buffer [50 mM Tris,
pH 8.0, 1 mM ADP, 2 mM glucose, 100 µM NADP+, 10 mM
MgCl2, 5 mM dithiothreitol, 0.02% BSA (wt/vol), 3 U/ml hexokinase (Roche Diagnostics), and 0.35 U/ml glucose-6-phosphate dehydrogenase (Roche Diagnostics)]. Production of NADPH,H+
was recorded after addition of creatine phosphate (24 mM). For citrate
synthase activity (CS, EC 4.1.3.7), 10 µl of protein extract (1:30
dilution, vol/vol) were added to 980 µl of assay buffer [100 mM
Tris, pH 8.0, 2 mM EDTA, 1.25 mM L-malate, 0.25 mM
NAD+, 0.01% Triton X-100 (vol/vol), 6 U/ml malate
dehydrogenase (Sigma)]. Production of NADH,H+ was recorded
after addition of acetyl-CoA (50 µM). For 3-hydroxyacyl-CoA dehydrogenase activity (HAD, EC 1.1.1.35), 3 µl of protein extract were added to 982 µl of assay buffer [50 mM Tris, pH 7.6, 2 mM EDTA,
50 µM NADH,H+, 0.01% Triton X-100 (vol/vol)]. Oxidation
of NADH,H+ was recorded after addition of acetoacetyl-CoA
(75 µM). For phosphofructokinase activity (PFK, EC 2.7.1.11), 10 µl
of protein extract (1:100 dilution, vol/vol) were added to 980 µl of
assay buffer [50 mM Tris, pH 8.0, 50 µM NADH,H+, 1.25 mM
ATP, 5 mM MgCl2, 2 mM
-mercaptoethanol, 0.05% BSA
(wt/vol), 0.45 U/ml aldolase (Roche Diagnostics), 50 U/ml triose
phosphate isomerase (Sigma Chemical), and 0.85 U/ml
glycerol-3-phosphate dehydrogenase (Roche Diagnostics)]. Oxidation of
NADH,H+ was recorded after addition of fructose 6-phosphate
(1 mM). For lactate dehydrogenase activity (LDH, EC 1.1.1.27), 5 µl
of protein extract (1:30 dilution, vol/vol) were added to 985 µl of
assay buffer (50 mM Tris, pH 7.6, 2 mM EDTA, and 50 µM
NADH,H+). Oxidation of NADH,H+ was recorded
after addition of pyruvate (1 mM).
Isolation of total RNA and RT-PCR conditions. Total RNA from frozen powdered muscles (30-40 mg) was isolated as previously described (11). One microgram of total RNA was used in reverse transcription reactions performed with a Superscript II kit (GIBCO-BRL) with the use of random hexamer primers at 42°C for 50 min. The reverse transcriptase was then inactivated for 5 min at 95°C. Primers homologous to nucleotides 2182 to 2203 (reverse: 5'-GTTTCATTACCTACCGTTATAC-3') and 2441 to 2460 (sense: 3'-CTCCTACTGTCTCCCTACCG-5') of PGC-1 were used. PCR amplification was performed with the LightCycler System (Roche Diagnostics) by using 10 µl of the reverse transcription reaction products diluted 1:20 (vol/vol) and the LightCycler-DNA Master SYBR Green I kit. The reaction mixture was first denatured for 2 min at 95°C and then cycled 40 times with a 55°C annealing for 10 s, a 72°C extension for 10 s, and a progressive denaturation to 95°C. Fluorescence was acquired during the extension period, and data were analyzed using the LightCycler analysis software.
Mitochondrial isolation and respiration.
TA muscle (100-120 mg) was washed in buffer A (50 mM
Tris buffer, pH 7.4, 100 mM KCl, and 5 mM EDTA), minced in buffer
A supplemented with 1 mM ATP (buffer B) (1:5, wt/vol),
and incubated with Nagarse (1 U/mg of muscle; Sigma Chemical) for 5 min. Digestion was stopped by addition of buffer B (1:30,
wt/vol). Muscle was then homogenized 16 × 2 s in a tissue
grinder (Thomas, Philadelphia, PA) at 1,500 rpm and centrifuged (5,000 g × 10 min). The pellet was resuspended in
buffer B (1:35, wt/vol) and centrifuged (800 g × 10 min). The supernatant was recovered. The
remaining pellet was resuspended in buffer B (1:35, wt/vol)
and centrifuged as above. Both supernatants were then combined and
centrifuged (9,000 g × 10 min). The resulting pellet
was suspended in buffer B (1:10, wt/vol) and centrifuged (9,000 g × 10 min). The final mitochondrial pellet was
diluted (1:1, wt/vol) in 10 mM Tris, pH 7.4, 1 mM EDTA, 700 mM
mannitol, and 220 mM saccharose solution. Protein content was measured
as described above. Oxygen consumption (natom
O · min1 · g
1 of muscle)
was recorded at 37°C under constant magnetic stirring with a Clarke
oxygen electrode (Hansatech, UK). State 4 rate of respiration was
measured in the presence of 100 µg of protein, 2.5 mM
L-malate, and 5 mM pyruvate in 1 ml of respiration medium (20 mM Tris, pH 7.4, 225 mM mannitol, 5 mM MgCl2, 500 µM
EDTA, 10 mM KH2PO4) saturated with air. State 3 rate of respiration was measured after the addition of ADP (350 µM).
To assess intactness of mitochondria, CS activity was measured in the
absence and presence of 0.01% Triton X-100 (vol/vol) as previously
described (13).
Statistical analysis. All values are presented as means ± SE. Two-way ANOVA was used to evaluate the effects of bupivacaine as a function of time on desmin protein level, state 3 rate of respiration, and CK, CS, HAD, PFK, and LDH enzyme activities. Post hoc comparisons were performed with the Fisher's protected least significance difference test. One-way ANOVA was used to evaluate the time course changes in necrosis, number of fibers with central myonuclei, PCNA and mtTFA protein levels, and PGC-1 mRNA level. Individual means were compared with a paired t-test. Differences were considered to be statistically significant at the 0.05 level of confidence.
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RESULTS |
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Body mass, muscle weight, and total protein concentration.
Bupivacaine injection had no effect on the weight of control muscles
over the 35-day time period (Table 1). At
the end of the experimental period, control muscle weight was
868.1 ± 54.8 mg (n = 5). Muscle weight was
significantly decreased (40%) by 5-10 days after bupivacaine
injection and had returned to control levels by 21 days postinjection.
The growth of animals did not affect total protein concentration of
control muscles, which averaged 168.4 ± 4.3 mg protein/g of
muscle (n = 44). Total protein concentration was
moderately decreased by 3-5 days postinjection (P < 0.05) and had recovered to control levels at day 7 (Table
1).
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Histochemical analyses of necrosis and regeneration.
No signs of necrosis were evident in control muscles. Normal fibers
were characterized by regular outlines and peripherally located nuclei.
Three days after bupivacaine injection, 99 ± 1% of the whole
muscle cross-sectional area was affected by the drug (Fig.
1, Ab and B). At
that time, most of the muscle fibers were disjointed or no longer
discernable. Intense myophagy with interstitial edema and infiltrating
cells was also observed. Proliferation and fusion of myoblasts occurred
concomitantly with the phagocytosis of muscle debris, since the
presence of many small colonies of regenerating fibers with central
myonuclei was observed as soon as 7 days postinjection (Fig.
1C). Massive reduction in myonecrosis was then observed
after 10 days of regeneration (P < 0.001; Fig. 1, Ac and B). At that time, the regenerating
myofibers had grown larger, and 52.2 ± 9.9% of the muscle fibers
had central myonuclei (Fig. 1, Ac and C). On
day 35, despite the presence of numerous fibers with central
myonuclei, muscle regeneration appeared to be stable (Fig.
1Ad). The normal polyhedric aspect of muscle fibers was
restored, and the number of muscle fibers was virtually the same in
controls (8,228 ± 807 fibers; n = 4) and
bupivacaine-injected TA muscles (7,640 ± 1,372 fibers;
n = 4).
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PCNA, desmin, and CPK protein levels.
To more closely delineate the period corresponding to myoblast
proliferation, PCNA, a protein that functions as a cofactor for DNA
polymerase- in S phase, was quantified by immunoblotting (Fig.
2, A and C).
Whereas PCNA was not detected in control muscles, regenerating muscles
strongly expressed PCNA. Expression peaked on day 3 and then
decreased to represent only 7% of day 3 values by 14 days
postinjection (14.5-fold decrease; P < 0.001). To
assess the onset of muscle-specific protein expression, desmin protein level was determined (Fig. 2, B and C).
Precocious expression of desmin is characteristic of proliferating
myoblasts (41, 45). A nonsignificant, 2.3-fold decrease in
desmin protein level (P = 0.11) was observed at 3 days
postinjection. Expression then increased, attaining levels that were
2.3-fold above those observed in control muscles on day 10 (P < 0.05). Desmin protein level had returned to
control levels at 35 days after bupivacaine injection. CPK is a
myogenic marker expressed after the fusion of myoblasts into myotubes
(9). Total CPK activity, whose 95% is due to the muscle
isoform (1, 2), exhibited a dramatic 80% reduction at 3 days of regeneration (P < 0.001; Fig. 2C)
and then gradually recovered, returning to control values 35 days after
bupivacaine injection.
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CS and HAD enzyme activities.
Matrix mitochondrial enzymes (CS and HAD) were assayed to establish a
possible relationship between mitochondrial content and skeletal muscle
regeneration (Fig. 3). These
mitochondrial enzymes correlate closely with mitochondrial volume
fraction (8). CS activity decreased significantly by 80%
at 3 days after bupivacaine injection. Enzyme activity then increased
sharply between days 5 and 10. On day
14, CS activity had returned to control values. The pattern of
changes in HAD activity was similar to that observed in CS activity,
exhibiting the same strong rise between days 5 and
10.
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Mitochondrial respiration.
To further study the effect of skeletal muscle regeneration on
mitochondrial content and function, we isolated mitochondria from
control and regenerating muscles and measured the rates of respiration.
Protein yield of mitochondria isolated from control muscles did not
change over the 35-day time period (5.59 ± 0.41 µg protein/mg
of muscle; n = 41). By 3 days postinjection,
mitochondrial protein yield was significantly decreased fourfold in
regenerating muscles compared with control muscles (Table
2). After 14 days of regeneration,
mitochondrial yield had nearly returned to control values. Intactness
of mitochondria in the suspension was virtually the same in control
(88.3 ± 1.0%; n = 41) and bupivacaine-injected (88.6 ± 0.9%; n = 41) muscles over the
experimental period. Addition of cytochrome c and
NADH,H+ to the respiration medium did not increase state 3 rate of respiration, further indicating the intactness of mitochondrial
membranes (data not shown). The efficiency of the mitochondrial
isolation, expressed as the percentage of CS activity in the
mitochondrial suspension over total muscle CS activity
(3), was unchanged in control (32.4 ± 2.4%;
n = 41) and bupivacaine-injected (33.1 ± 2.8%;
n = 41) muscles. With the use of CS activity as a
reference, mitochondrial respiration was therefore expressed per muscle
mass (3, 13). Respiratory control ratios (RCR) indicated
that mitochondria isolated from control muscles were well coupled
(Table 2). In contrast, RCRs were dramatically decreased at 3 days
postinjection (7-fold relative to control; P < 0.001).
Control values were reached between days 14 and 21 of the experimental period. State 4 rates of respiration were
similar in control (0.65 ± 0.05 natom
O · min1 · g muscle
1;
n = 41) and bupivacaine-injected (0.52 ± 0.05 natom O · min
1 · g muscle
1;
n = 41) muscles over the course of the regeneration
process. By contrast, state 3 rates of respiration were only 12% of
their respective contralateral values by 3 days postinjection
(P < 0.01; Fig. 3). From 5 to 10 days, mitochondrial
respiration sharply increased to reach control levels at 21 days
postinjection. This pattern closely resembled the pattern obtained with
CS and HAD activities.
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mtTFA protein level and PGC-1 mRNA level.
In view of the effects of muscle regeneration on mitochondrial content
and function, mtTFA protein level was quantified (Fig. 4). Surprisingly, the changes in mtTFA
protein level relative to contralateral values were moderate. Only
day 7 values were significantly different from those of
contralateral muscles. However, relative mtTFA protein levels were
different over time, from being lower between days 3 and 7 to being nearly the same to control values
between days 10 and 35. mtTFA expression is
activated by PGC-1 (49). PGC-1 mRNA level exhibited a
strong decrease 3 days after bupivacaine injection and then increased
to peak on day 10 (5-fold increase; Fig. 4,
inset). On day 21, PGC-1 mRNA level had almost
returned to control levels.
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PFK and LDH enzyme activities.
PFK and LDH activities were decreased by ~50% at 3 days
postinjection (P < 0.01; Fig.
5). No significant changes in enzyme activities were evident until 21 days of regeneration. Activities had
returned to control levels by day 35. This pattern of
expression was significantly different from the one observed for
mitochondrial enzymes (two-way ANOVA; data not shown).
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DISCUSSION |
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The ability to alter mitochondrial content and function is an important adaptive response of skeletal muscle. We hypothesized that skeletal muscle regeneration, which recapitulates embryonic myogenesis, could stimulate mitochondrial biogenesis. To address this question, we chose the bupivacaine-induced muscle degeneration model. The results indicate that skeletal muscle regeneration is accompanied by a dramatic stimulation of mitochondrial biogenesis concomitant with the onset of muscle differentiation.
Histochemical and biochemical analyses were performed to delineate some of the cellular events occurring over the course of the regeneration process. In agreement with other studies (20, 37, 39), bupivacaine was highly effective in eliciting muscle degeneration. The appearance of myogenic mitosis, as demonstrated by the expression of PCNA, was regarded as the first sign of the initiation of regeneration (38). Progression of myogenesis was also associated with the onset of muscle-specific protein expression, as indicated by the expression of desmin, one of the earliest known muscle-specific structural proteins (6, 16). The appearance of myotubes with central myonuclei concomitant with the increase in total CPK activity clearly indicates that myoblast fusion was initiated as early as 7 days of regeneration. The subsequent growth of regenerating myofibers (size and number) revealed that terminal differentiation was almost complete at the end of the experimental period, despite the persistence of fibers with central myonuclei. All together, two main periods emerged from the histochemical analyses of necrosis and regeneration and the temporal expressions of PCNA, desmin and CPK: 1) activation of satellite cells and proliferation of myoblasts (days 3-14) and 2) differentiation of myoblasts into muscle fibers (days 5-35).
The novel finding in this study was that in vivo skeletal muscle
regeneration was accompanied by a marked stimulation of mitochondrial biogenesis. The most prominent changes in the yield of mitochondrial proteins, CS activity, and state 3 rate of respiration occurred between
days 5 and 10 of regeneration, indicating a
nearly fivefold increase in mitochondrial content (8, 42)
during the onset of muscle differentiation. Gene expression of
mitochondrial proteins depends on the existence of a set of specialized
transcription factors (see Ref. 25 for review). Expression
of mitochondrial genome as well as mtDNA replication is regulated by
mtTFA, a nuclear-encoded protein imported inside the mitochondrion
(35). Binding sites for NRF-1 and -2 are present in the
human mtTFA promoter and are important for mtTFA gene expression
(48). Furthermore, PGC-1 also coactivates NRF-1
transcriptional activity on mtTFA gene expression (49).
One surprising finding in the present study was the moderate changes in
mtTFA protein level compared with control values. This contrasts with
the large decrease in CS enzyme activity and mitochondrial respiration
observed on days 3 and 5. Therefore, one may
hypothesize that mtTFA protein levels observed on days 3, 5, and 7 of regeneration would be necessary mainly for the
replication of mtDNA. However, mtDNA replication also depends on
factors other than mtTFA, such as DNA polymerase-, mitochondrial
single-strand DNA-binding protein, or RNA mitochondrial processing
enzyme (35). Protein level measurements of these factors
would therefore be necessary to confirm the physiological significance
of elevated mtTFA protein level during the onset of regeneration. Later
during the regeneration process, PGC-1 mRNA and mtTFA protein levels
peaked on day 10, concurrently with the state 3 rate of
respiration, suggesting that the transcriptional activation by PGC-1
and mtTFA may be one of the mechanisms regulating mitochondrial
biogenesis in regenerating skeletal muscle. Other aspects may also be
important in the overall regulation of mitochondrial biogenesis, like
the transcriptional activation by other transcription factors, the
regulation of mRNA stability, and/or the regulation of the import
process (25). Overall, these different regulatory influences may determine the expression pattern of mitochondrial proteins observed during in vivo skeletal muscle regeneration.
Biogenesis of functional mitochondria also requires intact mitochondrial membranes to house the newly synthesized proteins and maintain an electrochemical proton gradient during respiration. In skeletal muscle, the resting oxygen consumption measured in the absence of ADP (state 4 respiratory rate) is mainly due to passive flux of protons through the inner mitochondrial membrane (29). Therefore, the state 4 respiratory rates reported in the present study indicate that the proton permeability of the inner mitochondrial membrane was unaffected by bupivacaine injection, supporting the concept that the gross lipid composition of mitochondrial membranes was fairly similar in control and bupivacaine-injected muscles (7). Taken together, these results suggest that mitochondrial proteins were synthesized and imported in mature lipid bilayers between days 5 and 14 to give functional, well-coupled mitochondria.
The signals involved in the stimulation of mitochondrial biogenesis during muscle regeneration remain to be determined, but they most likely result from the combinatorial interplay of different molecular events. For example, the production of growth factors and cytokines by infiltrating cells and muscle cells (27, 33, 34) could modulate mitochondrial gene expression during skeletal muscle regeneration. Indeed, evidence has been recently presented for the role of growth factors in inducing cytochrome c gene expression through the modulation of cAMP response element-binding protein and NRF-1 transcriptional activity in BALB/3T3 cells (24). Calcium is also known to modulate mitochondrial gene expression (15) (D. Freyssenet, I. Irrcher, M. K. Connor, M. DiCarlo, and D.A. Hood, unpublished observations) as well as mitochondrial-nuclear cross talk (5) in muscle cells. With the modification of structural and functional characteristics of calcium homeostasis during skeletal muscle regeneration (40) taken into account, perturbations in cytosolic calcium concentration could also contribute to mitochondrial gene expression. Interestingly, calcium modulates muscle-specific gene expression and CS activity through a calcineurin-dependent pathway (4), therefore opening the possibility that a shared signal could regulate the expression of both mitochondrial and muscle-specific genes during skeletal muscle regeneration.
A new and potentially important observation has emerged from this study, an observation that establishes possible relationships between mitochondrial biogenesis and muscle cell differentiation. The different response of glycolytic and mitochondrial metabolisms observed in the present study suggests that the regenerating capability of rat skeletal muscle could depend on the efficiency of oxidative phosphorylation. Indeed, the onset of muscle differentiation was accompanied by the recovery of state 3 rate of respiration. This suggests that ATP production by mitochondria could regulate the execution of a number of regulatory and biosynthetic events involved in myogenesis. Furthermore, a recent report (43) demonstrates that mitochondria can also regulate in vitro muscle differentiation through the regulation of myogenic transcription factor expression. Together, these data corroborate previous studies showing that inhibition of mitochondrial protein synthesis inhibits in vitro muscle cell differentiation (22, 23, 28). Experiments are in progress to further explore the function of mitochondria during myogenesis.
In summary, we have shown that skeletal muscle regeneration is accompanied by a marked stimulation of mitochondrial biogenesis concomitant with the onset of muscle cell differentiation. This work sets the stage for in vivo studies designed to evaluate the physiological function of mitochondria in regulating skeletal muscle cell proliferation and differentiation.
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ACKNOWLEDGEMENTS |
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The rat mtTFA antibody was originally provided by Dr. H. Inagaki, National Industrial Research Institute, Nagoya, Japan. We thank Dr. P. Berthon (Université de Rennes), Dr. T. Busso, A. Barani, and A.-C. Durieux for their helpful comments on the manuscript. The technical assistance of J. Castells, Dr. G. Stépien (Centre Hospitalier Universitaire, Angers, France), and the Laboratoire de Biochimie et Biologie du Tissu Osseux (Faculté de Médecine, St-Etienne, France) is also acknowledged. Thanks are also due to Y. Boyer for animal care.
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FOOTNOTES |
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S. Duguez is a recipient of a grant from the Laboratoire de Physiologie, Groupe PPEH GIP E2S.
Address for reprint requests and other correspondence: D. Freyssenet, Laboratoire de Physiologie, Groupe PPEH GIP-E2S, Faculté de Médecine, 15 rue Ambroise Paré, 42023 St-Etienne, France (E-mail: damien.freyssenet{at}univ-st-etienne.fr).
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.
10.1152/ajpendo.00343.2001
Received 27 July 2001; accepted in final form 9 November 2001.
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