Influence of overload on phenotypic remodeling in regenerated skeletal muscle

A. X. Bigard1, J. Zoll1, F. Ribera1, P. Mateo2, H. Sanchez1, B. Serrurier1, and R. Ventura-Clapier2

1 Unité de Bioénergétique et Environnement, Centre de Recherches du Service de Santé des Armées, La Tronche; and 2 Cardiologie Cellulaire et Moléculaire, Unité 446 Institut National de la Santé et de la Recherche Médicale, Faculté de Pharmacie, Université Paris-Sud, Chatenay-Malabry, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We studied the effects of 10 wk of functional overload on the expression of myosin heavy chain (MHC), sarcoplasmic reticulum Ca2+-ATPase isoforms (SERCA), and the activity of several metabolic enzymes in sham and regenerated plantaris muscles. Overload was accomplished by bilateral surgical ablation of its synergists 4 wk after right plantaris muscles regenerated after myotoxic infiltration. The overload-induced muscle enlargement was slightly less in regenerated than in sham muscles [28% (P < 0.005) and 43% (P < 0.001), respectively]. Overload led to an increase in type I MHC expression (P < 0.01) to a similar extent in sham and regenerated plantaris, while the expected shift from type IIb to type IIa MHC was less marked in regenerated than in sham plantaris. The overload-induced decrease in the expression of the fast SERCA isoform and in the activity of the M subunit of lactate dehydrogenase occurred to a similar extent in sham and regenerated plantaris [66% (P < 0.01) and 27% (P < 0.005), respectively]. In conclusion, the lesser responses of muscle mass and fast MHC composition of regenerated plantaris to mechanical overload suggest an alteration of the transcriptional, translational, and/or posttranslational control of gene expression in regenerated muscle.

compensatory overload; myosin heavy chain; creatine kinase; lactate dehydrogenase; SERCA


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IN MAMMALS, ADULT MUSCLE FIBERS originate from at least two populations of myotubes that mature into adult myofibers (24). During fetal development, a population of myogenic cells gives rise to the mononucleate satellite cells (21). These myogenic cells are normally quiescent and located within the basal lamina of adult fibers. They are activated during muscle growth (2) or after muscle damage (1). Inasmuch as skeletal muscle fibers are repeatedly damaged and repaired throughout life, after various events such as diseases, trauma, injury, or mechanical stress (10), adult muscle comprises fibers that arise from several sets of myogenic cells, including satellite cells. The responses of regenerated myofibers to factors known to affect morphological, biochemical, and molecular characteristics of muscle are thus of functional relevance.

Muscle mass and muscle phenotype are determinants of contractile performance such as maximal power output, speed of shortening, and resistance to fatigue. Stretch and force generation are major factors of muscle enlargement (16), while contractile phenotype, mainly determined by the expression of myosin heavy chain (MHC) isoforms in skeletal muscle, is under the control of external factors such as neuronal influences, thyroid hormones, and increased contractile and weight-bearing activity (for review see Ref. 26). In addition, intrinsic factors, such as the developmental heterogeneity of myofibers, have been shown to play a role in the phenotypic response to changes in functional load. Previous studies demonstrated that regenerated muscles comprising a homogeneous population of fibers derived from satellite cells have a greater plasticity than normal muscles to endurance training (4, 5). Compensatory overload by ablation of synergists is a model of increased levels of contractile activity and mechanical stress (7, 15). This model is known to result in increased muscle mass and fiber hypertrophy as well as in a shift toward an increased expression of slower MHC isoforms (12, 13, 18, 23, 28, 31). Increasing mechanical load during the early phase of regeneration did not affect the relative composition of MHC in soleus muscle grafts (14). Whether muscle mass and MHC expression of fast-twitch muscle respond predictably to compensatory overload late after muscle injury has not been examined.

Transitions in cellular expression of MHC isoforms need to be coordinated with the expression of other functional components of muscle such as proteins involved in sarcoplasmic reticulum Ca2+ handling, sarcoplasmic reticulum Ca2+-ATPases (SERCA), and enzymes of energy metabolism. Two isoforms of SERCA are encoded by two distinct genes in skeletal muscle: SERCA1 in fast-twitch fibers and SERCA2a in slow-twitch fibers (9). Chronic overload of sham plantaris muscle by synergistic muscle ablation results in upregulation of the SERCA2a gene with a high degree of coordination of expression between MHC and SERCA isoforms in single fibers (17, 30). Nevertheless, the extent and direction of overload-induced adaptations of creatine kinase (CK) and lactate dehydrogenase (LDH) isozymes, two important families of metabolic enzymes contributing to the molecular diversity of muscle fibers, and their coordination with the shift in MHC isoforms in sham fast-twitch muscles need to be determined.

As in sham muscles, changes in MHC isoforms need to be coordinated with associated changes in metabolic enzymes and SERCA isoforms in regenerating skeletal muscles. We previously showed that, in contrast to alterations in the relative distribution of MHC isoforms in regenerated muscle, the training-induced changes in citrate synthase (CS) activity and CK and LDH isozyme distribution occurred to a similar extent in sham and regenerated fast-twitch muscles (5). In light of this finding, it was interesting to examine the coordinated response of contractile and metabolic proteins to a model of hypertrophy in regenerated muscle.

Therefore, the aim of this study is a better understanding of the effects of increased mechanical load on muscle mass and expression of several proteins that account for contractile and metabolic properties in regenerated fast-twitch muscle. We investigated 1) whether the mass of regenerated muscle was affected by compensatory overload and 2) whether there was a coordinated expression of proteins involved in contraction (i.e., MHC isoforms), relaxation (i.e., SERCA isoforms), and enzymes of energy metabolism (i.e., CS activity and CK and LDH isozyme distribution) in sham and regenerated muscles. This issue is important from a fundamental and a physiopathological point of view, since it concerns the capacity of regenerated muscle to respond to external factors such as load and contractile activity. Degeneration of the right plantaris muscle was first induced by injection of a myotoxic snake venom, a well-defined model of muscle injury known to induce a rapid regeneration of myofibers (19). Because myotoxic-induced regeneration gives rise to a muscle comprising fibers arising synchronously from only one population of satellite cells, this model is valid for studying the response of newly formed myofibers to mechanical overload.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Experimental protocol and surgical procedures. Male rats of a Wistar strain, initially weighing ~60 g, were purchased from IFFA Credo (L'Arbresle, France). Animals were housed four per cage in a thermoneutral environment (22 ± 2°C), on a 12:12-h photoperiod, and were provided with food and water ad libitum. This investigation was carried out in accordance with the Helsinki Accords for Humane Treatment of Animals During Experimentation. They were assigned to a control (C, n = 6) or an overloaded (OV) group (n = 6). Because the aim of this study was to examine the response of fully regenerated plantaris muscle to overload, a third group of rats was constituted to determine the characteristics of regenerated plantaris muscles just before the surgical ablation of its synergists, 4 wk after muscle injury (iC, n = 6).

After 5 days of acclimatization to the animal room, rats were anesthetized with pentobarbital sodium (40 mg/kg), and myofiber degeneration was induced by injection of Naja nigricollis snake venom into the belly of right plantaris muscles, as previously described (5). Among the suitable models, myotoxic injections give rise to a regenerative process that takes place earlier and develops more rapidly than in the case of other models of muscle injury (19). After skin incision under aseptic conditions, a 25-gauge, 5/8 (0.5 × 16-mm) needle was inserted near the distal tendon, and 0.3 ml of venom from the N. nigricollis snake [cardiotoxin, 10 µg/ml in 0.9% (wt/vol) NaCl solution] was injected slowly by a single injection into the plantaris muscle (Latoxan Rosans). The same skin incisions and muscle manipulations were then performed on left hindlimbs. The needle was inserted into the belly of left plantaris muscles without injection of venom or vehicle.

Animals of the OV group were anesthetized by an injection of pentobarbital sodium (40 mg/kg ip) 4 wk after induction of degeneration in the right plantaris. Compensatory overload of plantaris was accomplished by the bilateral removal of its synergists, as previously described (3). The dorsal muscles of both legs were exposed, and the major portion of the gastrocnemius was excised after a blunt dissection, while the soleus muscle was entirely excised. Care was taken to ensure that the blood and nerve supplies to the plantaris were not affected. Sham operations were routinely performed in C rats.

Tissue preparation. At 4 wk after cardiotoxin infiltration (iC group) and 10 wk after surgical treatment (OV and C groups), rats were anesthetized with pentobarbital sodium (80 mg/kg body wt). To verify that the regenerated neuromuscular junctions revealed no apparent functional denervation and that the nerve remained intact after surgical ablation of the synergist muscles, the right sciatic nerve was stimulated before dissection of the plantaris muscle. Right (i.e., injected with the toxin) and left (i.e., non-venom-treated) plantaris muscles were then excised, cleaned of adipose and connective tissue, and weighed wet. The distal parts of the plantaris muscles were quickly frozen in liquid nitrogen, while the proximal parts were mounted in an embedding medium (TEK OCT compound) and frozen in isopentane cooled to the freezing point (-160°C) by liquid nitrogen. All samples were stored at -80°C until histochemical and biochemical analyses were performed.

Histology and immunocytochemistry. Serial transverse sections (10 µm thick) were cut from the midbelly portion in a cryostat maintained at -20°C and stained with hematoxylin and eosin. Five mouse monoclonal antibodies directed against specific MHC isoforms were used in this study: antibody reacting with slow type I (Novocastra, reference NCL-MHCS, Newcastle Upon Tyne, UK), with all adult fast and developmentally regulated epitopes but not with slow myosin (MY-32, Sigma Chemical, St. Louis, MO), with fast type IIa (SC-71) or fast type IIb MHC isoforms (BF-F3), or with all isoforms except fast type IIx (BF-35). SC-71, BF-F3, and BF-35 antibodies have been previously characterized (25). In addition, sections were incubated with a monoclonal antibody recognizing embryonic and neonatal MHC isoforms but not adult isoforms (RNMY2/9D2, Novocastra). For immunohistochemical detection of SERCA isoforms, serial cross sections were incubated with primary antibodies specific for SERCA1 and SERCA2a (V12G9 and IID8, respectively, Novocastra). As previously described, the avidin-biotin immunohistochemical procedure was used for the localization of the antigen-antibody binding (Vector Laboratories, Burlingame, CA) (5).

For all muscles, a sample of ~400 fibers that were free from artifact were randomly selected from fields equally distributed over the biopsy for single-fiber MHC composition. Fibers were classified according to their staining profile with the aid of a microscope linked to a computer-based image analysis system (Visiolab 200, Nikon-France). Fibers demonstrating a positive reaction to a specific antibody were considered to be expressing that specific MHC isoform. Negative control sections with omission of the primary antibodies were randomly included in the immunostaining procedures.

Analysis of MHCs. Plantaris muscles were subjected to the analysis of MHC isoforms by use of techniques described previously (29). Tissue samples were minced with scissors in 9 vol of a solution containing 20 mM NaCl, 5 mM sodium phosphate, and 1 mM EGTA (pH 6.5). Myosin was then extracted with 3 vol of 100 mM sodium pyrophosphate, 5 mM EGTA, and 1 mM dithiothreitol (pH 8.5). After 30 min of gentle shaking, the mixture was centrifuged at 12,000 g for 10 min, and the supernatant containing myosin was diluted with 1 vol of glycerol and stored at -20°C. The separating gel solution contained 30% glycerol, 8% acrylamide-bis (50:1), 0.2 M tris(hydroxymethyl)aminomethane (Tris), 0.1 M glycine, and 0.4% sodium dodecyl sulfate (SDS). The composition of the stacking gel was 30% glycerol, 4% acrylamide-bis (50:1), 70 mM Tris, 4 mM EDTA, and 0.4% SDS. Myofibril samples were denatured by using a sample buffer containing 5% beta -mercaptoethanol, 100 mM Tris-base, 5% glycerol, 4% SDS, and bromphenol blue. Electrophoresis was performed using a Mini Protean II system (Bio-Rad Laboratories, Hercules, CA). Gels were run at constant voltage (70 V) for ~28 h and then stained with Coomassie blue. The MHC protein isoform bands were scanned and quantified by using a densitometer system equipped with an integrator (model GS-700, Bio-Rad Laboratories).

SERCA1 Western blot analysis. Frozen muscle samples were minced and then processed with a glass homogenizer in a buffer containing (in mM) 50 Tris · HCl (pH 7.5), 100 NaCl, 2% (wt/vol) SDS, and 0.2 phenylmethylsulfonyl fluoride. The homogenates were centrifuged at 1,000 g for 10 min at 4°C, and the supernatant was withdrawn and stored on ice. Protein concentrations were determined by the method of Lowry et al. (21). Equal amounts of protein (5 µg/lane) were separated on 1-mm-thick 10% SDS-polyacrylamide electrophoresis gels. Proteins were then transferred to polyvinylidene difluoride membranes (Immobilon, Millipore, Bedford, MA) in a high-intensity (60 V) electrical field for 2 h with use of platinum-coated plate electrodes (Trans-Blot Cell, Bio-Rad) at 4°C. Blots were blocked for nonspecific protein binding with 5% (wt/vol) nonfat milk for 2 h and then incubated overnight (4°C) with a monoclonal antibody against the fast Ca2+ pump SERCA1 (V12G9, Novocastra). Primary antibody binding was detected with an enhanced chemiluminescence Western blotting kit (Amersham) according to the manufacturer's instructions. Band intensity was quantified by laser densitometry (model GS-700, Bio-Rad Laboratories).

CK, CS, and LDH activities. Frozen tissue samples were weighed and placed into an ice-cold homogenization buffer (30 mg wet wt/ml) containing 5 mM HEPES (pH 8.7), 1 mM EGTA, 1 mM dithiothreitol, 5 mM MgCl2, and 0.1% Triton. Samples were processed using a micro-glass hand homogenizer and incubated for 60 min at 0°C to ensure complete enzyme extraction. Whole tissue homogenate was used for the determination of specific activity and isozyme fractionation.

CS activity was determined according to Srere (27). The total activities of adenylate kinase (AK) and CK were assayed in a solution containing (in mM) 20 HEPES, 5 MgCl2, 20 glucose, 1.2 ADP, and 0.6 NADP, pH 7.4, at 30°C. AK activity was determined by measuring the production of NAPDH from a glucose-6-phosphate dehydrogenase/hexokinase (2 IU/ml)-coupled enzyme reaction in the absence of phosphocreatine (PCr). CK total activity was determined by subtraction, after addition of PCr (20 mM). NADPH production was measured spectrophotometrically at 340 nm (UVIKON spectrophotometer, Kontron Instruments, Milan, Italy).

CK isozymes were separated using agarose (1%) gel electrophoresis performed at 200 V for 90 min. Internal standards of commercial (MM) CK were run in parallel with tissue samples to ensure linearity in isozyme quantification. To avoid saturation of the various CK isoforms, three dilutions were used for each sample. Individual isozymes were resolved by incubating the gel with a paper soaked with staining solution for 40 min in the dark at room temperature. The staining solution contained (in mM) 22 MES (pH 7.4), 50 magnesium acetate, 79 glucose, 120 N-acetylcysteine, 9 ADP, 120 PCr, 9 NADP, and 0.1 P1,P5-di(adenosine-5')-pentaphosphate (to inhibit AK), hexokinase (9 IU/ml), and glucose-6-phosphate dehydrogenase (6 IU/ml). Isozyme bands were visualized by the fluorescence of NADPH and quantified using an image analysis system (Bio-Rad Laboratories). The LDH isozyme profile was determined using agarose gel electrophoresis (LDH reagent kit, Sigma Chemical) at 200 V for 90 min, followed by image analysis. Activity of CK and LDH isozymes was quantified by multiplying each percentage by total activity, as determined spectrophotometrically.

Statistical procedures. All data are presented as means ± SE. Data were analyzed with two-way analysis of variance with compensatory overload and regeneration as the two main factors. The main statistical effects of compensatory overload and regeneration were detected on the experimental variables, as well as the interaction effect between these two factors. When appropriate, differences between groups were tested with a Newman-Keuls post hoc test. Statistical significance was accepted at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Body and muscle weight. The mean body weight of OV rats was unchanged relative to C animals (Table 1). The absolute weight of sham and regenerated plantaris muscles of the OV group increased by 34% (P < 0.01) and 21% (P < 0.05) relative to their respective control muscles. The normalized weight (mg/g body wt) of sham plantaris muscle was 43% greater in the OV than in the C group (P < 0.001), whereas it was only 28% greater in regenerated muscles (P < 0.005).

                              
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Table 1.   Morphological data in experimental groups

Characteristics of regenerated plantaris muscle before compensatory overload. Hematoxylin and eosin staining showed that the majority of fibers in regenerated plantaris in the iC group was characterized by centrally located nuclei. Fibers were homogeneous in size, with a polygonal shape. None of the measured contractile or metabolic parameters of regenerated plantaris differed from those of sham muscles (Table 2). Four weeks after injection of cardiotoxin into the right plantaris muscle, the central location of the nuclei remained the main feature distinguishing regenerated from nonregenerated fibers. It can be concluded that regenerated and sham plantaris have similar markers of metabolic and contractile phenotypes just before surgical removal of synergist muscles.

                              
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Table 2.   Characteristics of plantaris muscle 4 wk after myotoxin treatment (iC group)

MHC distribution in plantaris muscles. As expected, overload induced a marked shift from fast to slow MHC isoforms (Fig. 1). The percentage of type I MHC in sham plantaris muscles of OV rats was four times that in sham muscles of C animals (P < 0.001). Moreover, there was a global effect of compensatory overload that resulted in an increase in the relative content of type IIa MHC (P < 0.05) and a marked decrease in the content of type IIb MHC (P < 0.001). The overload-induced increase in the proportion of type I MHC was similar in sham and regenerated plantaris (P < 0.01). A two-way interaction between compensatory overload and regeneration was shown for the proportion of type IIa MHC (P < 0.001). As a result, the overload increase in the percentage of type IIa MHC was observed only in sham plantaris (63%, P < 0.01). On the other hand, the decrease in the proportion of type IIb MHC was more marked in sham than in regenerated plantaris [-48% (P < 0.01) and -28% (P < 0.05), respectively]. This lesser effect of overload in the response of regenerated muscles was expressed by a lower content of type IIa MHC (-42%, P < 0.05) and a higher relative content of type IIb MHC in regenerated than in sham muscles (44%, P < 0.05).


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Fig. 1.   Representative SDS-polyacrylamide gel for separation of adult myosin heavy chain (MHC) isoforms and MHC distribution in sham and regenerated (reg) plantaris muscles from control rats (C) and rats overloaded by bilateral removal of the synergistic gastrocnemius and soleus muscles (OV). a, Sham plantaris muscle of C group (shamC); b, regenerated plantaris muscle of C group (regC); c, sham plantaris muscle of OV group (shamOV); d, regenerated plantaris muscle of OV group (regOV). Values are means ± SE. Significantly different from corresponding muscle in C group: *P < 0.05; **P < 0.01. Significantly different from sham muscle: $P < 0.05. In top, note 1) overload-induced increase in type I MHC in sham and regenerated muscles and 2) less overload-induced decrease in type IIb MHC and increase in type IIa MHC in regenerated than in sham muscle of OV group.

Expression of the SERCA1 protein by immunoblotting analysis. The expression of the fast-specific SERCA isoform SERCA1 is markedly decreased by compensatory overload (P < 0.001). The mean intensity of the signal from SERCA1 protein decreased by 67% in sham muscle as a result of synergist ablation (P < 0.01; Fig. 2). The overload-induced decrease in SERCA1 protein was similar in regenerated plantaris muscle (-66%, P < 0.01).


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Fig. 2.   Representative Western blot and quantitative immunoblotting analysis of the fast-specific sarcoplasmic reticulum Ca2+-ATPase (SERCA) isoform in sham and regenerated plantaris (PLA) muscles from control and overloaded rats. Equal amounts of protein (5 µg) were loaded per lane on 1-mm-thick 10% SDS-polyacrylamide gels. Average signals of lanes were lower in OV than in C for sham and regenerated plantaris muscles. AU, arbitrary units after quantification by laser densitometry. a, Sham plantaris muscle of C group; b, regenerated plantaris muscle of C group; c, sham plantaris muscle of OV group; d, regenerated plantaris muscle of OV group. **Different from C values (P < 0.01). In top, note overload-induced decrease in the average signal in sham and regenerated muscles.

Immunohistochemical analysis of the expression of MHC and SERCA isoforms. Overload-induced changes in the fiber MHC isoform expression are reported in Fig. 3. In sham plantaris muscles of OV rats, the percentage of fibers displaying type I MHC was four times that of C animals (Fig. 4). The number of hybrid fibers containing type I and IIa MHC isoforms was only slightly increased in sham OV muscles compared with sham muscles of C rats (2.8 and 1.8%, respectively, not significant). A shift in the MHC profile was observed at the single-fiber level between the fast MHC isoforms. At 10 wk after ablation, the percentage of fibers containing type IIa MHC was twice that in C rats (P < 0.01), and only 11% of fibers expressed or coexpressed type IIb MHC. No reexpression of developmental MHC isoforms was detected in overloaded plantaris muscles. Nearly all fibers containing type I MHC labeled positively for SERCA2a, and, conversely, nearly all fibers with at least one fast MHC isoform were positive for SERCA1 (Fig. 4). A strong correlation was found between the percentage of fibers expressing type I MHC and those expressing SERCA2a (r = 0.92, P < 0.0001), as well as between the percentage of fibers expressing fast isoforms of MHC and those containing SERCA1 protein (r = 0.93, P < 0.0001). Moreover, fibers coexpressing SERCA1 and SERCA2a were also those coexpressing type I MHC and at least one of the fast MHC isoforms.


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Fig. 3.   Percentage of fibers expressing or coexpressing each major MHC isoform in sham and regenerated plantaris muscles of C and OV groups. Values are means ± SE. Fibers expressing type IIx MHC are pure type IIx fibers containing type IIx MHC alone. Significant difference between C and OV groups: *P < 0.05; ** P < 0.01. Significantly different from sham and regenerated muscles: $P < 0.05; $$ P < 0.01.



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Fig. 4.   Serial transverse sections of sham plantaris muscles from control (A-F) and overloaded rats (G-L) and of regenerated overloaded plantaris muscles (M-R). Myofibers were stained with antibodies raised against slow MHC (A, G, and M), type IIa MHC (B, H, and N), all MHC isoforms except fast type IIx (C, I, and O), type IIb MHC (D, J, and P), SERCA2a (E, K, and Q), and SERCA1 (F, L, and R). Note the lower percentage of fibers expressing type IIa MHC (N vs. H) and the higher percentage of fibers expressing type IIb MHC (P vs. J) in regenerated than in sham plantaris muscles after functional overload. Scale bar, 500 µm.

The percentage of fibers containing type I MHC was similar in regenerated and sham plantaris muscles as a result of overload. However, the overload-induced shift in the cellular expression of fast MHC isoforms on the order of IIb right-arrow IIx right-arrow IIa was less marked in regenerated than in sham muscles. The percentage of fibers expressing type IIa MHC was lower in regenerated plantaris muscle of OV rats than in sham muscles (P < 0.01; Fig. 4, N and H) and did not differ from that in regenerated muscles of C rats. Moreover, the proportion of type IIb fibers in regenerated plantaris was higher in OV than in sham muscles (P < 0.05; Fig. 4, P and J). The proportion of hybrid fibers containing type I and II MHC isoforms was similar to that in sham muscles. In sham plantaris muscle, a high correlation was found between the percentage of fibers labeled positively for SERCA2a and the proportion of fibers containing type I MHC (r = 0.94, P < 0.0001). The antibody against embryonic and fetal MHCs gave undetectable staining in all cases (data not shown).

Total CS, CK activities, and CK isozyme distribution. A trend toward a global decrease in CS activity was shown after compensatory overload (P = 0.055; Table 3). No major change was shown in CS, CK activities, and CK isozyme distribution in sham plantaris as a result of overload. The responses of regenerated plantaris to overload were similar to those in sham muscles, only as statistical trends. In contrast to the iC group, the B subunit of CK was not detected in regenerated plantaris of the C or the OV group.

                              
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Table 3.   CS activity, CK isozyme profile, and specific activities in C and OV plantaris muscles

LDH isozyme profile. A global effect of overload was observed, resulting in a marked decrease in the total LDH activity (P < 0.001) in sham and regenerated muscles (28 and 33%, respectively, P < 0.005; Fig. 5). This decrease in total LDH activity in the OV group resulted mainly from a decrease in the specific activity of the M subunit in sham and regenerated plantaris (27%, P < 0.01).


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Fig. 5.   Total activity and specific activity of the H and M subunits of lactate dehydrogenase (LDH) in sham and regenerated plantaris muscles from control and overloaded rats. Values are means ± SE. Enzyme activities are expressed as units of activity per gram of tissue wet weight (IU/g). Significant difference between OV and C plantaris muscles: *P < 0.05; **P < 0.01.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The aim of this study was to assess the impact of increased mechanical load on muscle mass and responses of several markers of the contractile and metabolic phenotypes in regenerated muscle. The main findings were as follows. 1) The overload-induced increase in the mass of regenerated plantaris muscle was slightly less than in sham muscle. 2) The marked increase in type I MHC resulting from compensatory overload was similar in sham and regenerated plantaris. 3) The overload-induced transition between the fast MHC isoforms was more marked in sham than in regenerated plantaris muscles. 4) The relative decrease in fast MHCs was associated with a highly coordinated decrease in the content of SERCA1, the fast isoform of SERCA, whatever the fiber origin. 5) Despite no changes in oxidative markers [CS, H subunit of LDH, and the mitochondrial isoform of CK (Mi-CK)], the overload-induced fast-to-slow phenotype transition was associated with a decrease in the total and M subunit specific activity of LDH in sham and regenerated muscle.

Changes in muscle mass. The surgical removal of synergistic muscles is known to result in a compensatory hypertrophy of the remaining muscle (13, 18, 23, 28, 31). Our results show that the overload-induced skeletal muscle hypertrophy was less in regenerated than in sham plantaris muscle. Previous studies demonstrated that increased mechanical load by ablation of synergistic muscles enhanced growth of regenerating muscles (14). However, whether the overload-induced muscle enlargement differed in regenerated and sham muscles has not been examined. Muscle hypertrophy is mainly controlled at the level of translation by the number of ribosomes at the onset of mechanical overload and, thereafter, by an increase in the transcription rate (8, 20). Whether overload induced less increase in ribosomal RNA and/or myofibrillar protein mRNA in regenerated than in sham plantaris muscles remains to be examined in future studies. Muscle responses to functional demand are related to changes in muscle mass and protein quantities and also to alterations in muscle phenotype expressed by changes in the content of protein isoforms and metabolic enzymes.

Response of MHC isoforms to overload. The overload-induced shift in MHC expression from the fastest (type IIb) to the slowest (type I) MHC isoform observed in the present study in sham plantaris muscle at the whole muscle level, as well as at the single-fiber level, is consistent with previous studies (13, 28). It has been previously shown that regenerated muscles demonstrate greater plasticity of myosin isozyme expression than did sham muscles in response to changes in functional load (4, 5, 11, 12). In contrast, the results of the present study showed that while the response of type I MHC was similar in sham and regenerated plantaris, increased mechanical stress had less effect on the fast MHC profile of regenerated plantaris. The overload-induced increase in the percentage of type IIa MHC was shown only in sham plantaris, and the percentage of type IIb MHC remained higher in regenerated than in sham muscles. Taken together, these results are an illustration of the lesser transition between fast MHC isoforms. In contrast to muscle enlargement, adaptive changes of contractile phenotype seem to be controlled at the level of transcription as much as at posttranscription.

SERCA expression in sham and regenerated plantaris in response to overload. Together with myosin, SERCA proteins are, at least partly, responsible for contractile properties of striated muscles. Chronic overload is known to induce a fast-to-slow shift in the expression of the proteins involved in sarcoplasmic reticulum Ca2+ handling (SERCA) (17, 30). In the present study, immunoblotting analysis demonstrated a 67% decrease in the SERCA1 expression, and immunohistochemical analyses showed that the overload-induced fast-to-slow shift in MHC isoform was highly correlated with the increase in the percentage of fibers containing the slow SERCA isoform. These results are consistent with a high degree of coordination of expression of MHC isoforms and SERCA proteins in intact muscles in response to overload (6, 30). Moreover, the extent of the overload-induced decrease in the SERCA1 expression was similar in sham and regenerated plantaris. Because all fast-twitch fibers express the SERCA1 isoform, the lack of specific effect of regeneration on the response of SERCA expression to overload is consistent with the expression of total fast MHC isoforms.

Effects of overload on the activity of metabolic enzymes. Despite a fast-to-slow phenotype transition, previous studies showed that the activities of several oxidative enzymes are decreased in overloaded plantaris, while the substrate oxidation capacity is not affected (3, 23). The trend toward a decrease in CS activity and the lack of overload effect on activity of Mi-CK and H subunit of LDH shown in this study confirm these findings. However, the overload-induced decreases in total LDH activity and in the specific activity of the M subunit of LDH are consistent with a decreased participation of the anaerobic glycolytic pathway in muscle metabolism and a more efficient energy utilization (3). Changes in the activity of metabolic enzymes observed in regenerated plantaris were similar to those shown in sham muscles. Type IIa fibers are known to have a higher oxidative potential than other fiber types in rats (13), and there seems to be a discrepancy between the similar response of metabolic enzymes to overload in sham and regenerated muscles and the differential response of the MHC pattern expression. However, this finding is consistent with previous results that showed that, in contrast to MHC isoforms, regenerated muscles are as sensitive as sham muscles to biochemical adaptations in response to endurance training (5).

Differential responses to overload of muscle mass and MHC expression. Just before mechanical overload of the plantaris was induced, 4 wk after myotoxin treatment, many markers of maturation, such as muscle mass, the downregulation of developmental MHC isoforms, and the pattern of adult MHC isoforms, were similar in regenerated and sham plantaris muscles (Table 2). It is thus suggested that the differential responses of sham and regenerated muscles to overload were more related to specific responses of regenerated muscle than to alterations in growth rate and maturation. The responses of muscle mass and MHC phenotype to compensatory overload in regenerated muscles contrast with those resulting from endurance training (4, 5). This finding could be related to differences in models of increased functional load. Although treadmill running is a model of increased neuromuscular activity imposed during a limited period, the model of surgical ablation of synergistic muscles is a model that induces a 24-h passive increased tension on the remaining muscles (7). The lesser muscle enlargement and transition between fast MHC isoforms observed in regenerated plantaris in response to overload is difficult to explain. Future studies are needed to examine the transcriptional control of the gene encoding MHC isoforms, the translational capacity, and the posttranscriptional control in overloaded regenerated muscle.

In conclusion, the present data show that the expected muscle enlargement and shift in the fast isoforms in response to mechanical overload were less marked in regenerated than in sham muscles, showing an alteration in protein quantity and isoform expression. Thus the effects of overload on muscle mass and expression of fast MHC isoforms depend on the ontogenic origin of myofibers. Thus chronic increased mechanical loading is suggested to affect the transcriptional, translational, and/or posttranslational control of expression of different subsets of genes in regenerated muscle. In contrast, the responses of SERCA proteins and metabolic enzymes to overload did not differ in regenerated and sham muscles. As a consequence, the coordinated response of contractile and metabolic proteins to compensatory overload was altered in regenerated fast muscle.


    ACKNOWLEDGEMENTS

This research was generously supported by the Association Française contre les Myopathies.


    FOOTNOTES

Address for reprint requests and other correspondence: A. X. Bigard, Unité de Bioénergétique et Environnement, CRSSA, BP 87, 38702 La Tronche Cedex, France (E-mail: BIGARDXavier{at}compuserve.com).

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.

Received 4 December 2000; accepted in final form 28 June 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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