Differential modulation of myosin heavy chain phenotype in an inactive extensor and flexor muscle of adult rats

Abstract

The effects of chronic neuromuscular inactivity on the phenotype and size of muscle fibres in a fast ankle extensor (medial gastrocnemius, MG) and a fast ankle flexor (tibialis anterior, TA) muscle of the rat hindlimb were determined. Inactivity was produced by spinal cord isolation (SI), i.e. complete spinal cord transections at a mid-thoracic and high sacral level and bilateral deafferentation between the transection sites. After 90 days of SI, the MG and TA muscle weights were 53 and 45% lower than in age-matched controls. Overall mean fibre sizes in the deep (close to the bone) and superficial (away from the bone) regions were ∼60 and 65% smaller in the MG and ∼40 and 50% smaller in the TA of SI than control rats, respectively. The myosin heavy chain (MHC) composition shifted towards the faster isoforms after SI: the MG showed an increase in both types IIx (20%) and IIb (23%), whereas the TA showed a marked increase in type IIx (94%) and a decrease in type IIb (18%) MHC. Both muscles in SI rats showed no type IIa and only one MG muscle had ∼5% type I MHC. These results show that prolonged inactivity has a stronger effect on a fast extensor compared with a fast flexor in the rat hindlimb. The larger decrease in mass and fibre size in the MG than the TA most probably reflects the larger impact of chronic inactivity on the normally more highly recruited extensor than flexor muscle. The primary shift to type IIb MHC in the MG and type IIx MHC in the TA indicate a different default mode for an inactive extensor vs. flexor muscle, and may reflect differing activity-independent neural influences, i.e. neurotrophic factors, on muscle fibre phenotype in extensors vs. flexors.

Introduction

Neuromuscular activity, i.e. activation and loading, is an important determinant of skeletal muscle properties (Salmons & Henriksson, 1981; Pette & Vrbova, 1985; Roy et al. 1991; Michel et al. 1996; Staron et al. 1998). A decrease in neuromuscular activity, as experienced with hindlimb unloading, spaceflight or spinal cord injury, results in skeletal muscle atrophy and a general shift towards faster mechanical and metabolic properties (Roy et al. 1991), as reflected by adaptations in the myosin heavy chain (MHC) isoform profile (Talmadge et al. 1995; Talmadge, 2000). The relationship between the neuromuscular activity level and muscle adaptations, however, is confounded in these models of reduced activity because the residual amounts and patterns of neuromuscular activity are variable and difficult to quantify (Alaimo et al. 1984; Alford et al. 1987; Blewett & Elder, 1993). By contrast, inactivity models, such as spinal cord isolation (SI) and tetrodotoxin (TTX) administration, have a near zero baseline of neuromuscular activity, and thus provide a clear resolution of the level of activity-dependent modulation of muscle properties.

The time course of adaptations in fibre size and MHC composition in the soleus (Grossman et al. 1998) and medial gastrocnemius (MG) muscles (Roy et al. 2000) have been studied up to 60 days after SI. In addition, we have determined the extent of these adaptations in the soleus muscle after 90 days and found dramatic changes in the MHC profile between 60 and 90 days (Huey et al. 2001). In the present study, we have used the SI model to determine the effects of long-term inactivity on the size and MHC profile of predominantly fast muscles having different primary functions. We have focused on a fast ankle extensor (MG) and a fast ankle flexor (tibialis anterior, TA), because the size and mechanical properties are affected by chronic periods of reduced activity or inactivity to a greater degree in extensors than in flexors (Roy et al. 1991, 2005).

The primary purposes of the present study were to determine the effects of prolonged inactivity (90 days) on (1) the cross-sectional area (CSA) of fibres of a known myosin phenotype, and (2) the MHC isoform profile at the whole muscle (gel electrophoresis) and single fibre (immunohistochemistry) levels. Based on previous findings using this model of inactivity for shorter periods (Roy et al. 2000, 2005), we hypothesized that the adaptations would be more severe in the MG than TA and that the default mode for the MHC isoform would be different for the two muscles, i.e. type IIb for the MG and type IIx for the TA. Based on the results from the soleus muscle in SI rats (Huey et al. 2001), we further hypothesized that the adaptations in the MHC profiles would be dramatically different from those observed after 60 days of SI. These results allow us to compare the default state for maintaining cell size and the type of myosin expressed for two muscles that differ in function but are relatively similar phenotypically.

Methods

Animals and surgical procedures

Adult female Sprague–Dawley rats (242 ± 2 g body weight) were assigned randomly to a control (n = 7) or an SI (n = 8) group. The SI procedures are a modification of the original protocols of Tower (1937) and these procedures and the care for the SI rats have been detailed previously (Roy et al. 1992; Grossman et al. 1998). Briefly, the rats were anaesthetized with ketamine hydrochloride (100 mg kg⁻¹) and xylazine (5 mg kg⁻¹) administered i.p. Supplemental doses of ketamine (30% of the initial dose, i.p.) were given as needed. Under aseptic conditions, a longitudinal midline skin incision was made over the spinal column from the T6 to the L6 vertebral levels and a partial laminectomy was performed between vertebral levels T7 and L5. After opening the dura, the dorsal roots were cut subdurally bilaterally from the mid-thoracic spinal cord level to S1. Lidocaine hydrochloride (1%; 2–3 drops) was applied to the transection sites. The spinal cord was lifted gently with a curved probe or fine forceps and completely transected at both mid-thoracic and high sacral spinal cord levels using microdissection scissors. Complete spinal cord transections were verified by lifting the cut ends of the spinal cord and passing a glass probe along the vertebral wall through the transection sites. Gelfoam was packed between the cut ends of the spinal cord at each transection site. A strip of gelfilm was placed along the length of the exposed spinal cord. The paravertebral muscles and fascia surrounding the spinal column were sutured using 4-0 Chromic gut and the skin incision was closed using 4-0 Ethilon suture.

The rats were allowed to recover fully from anaesthesia in an incubator (37 °C) and were given lactated Ringer's solution (5 mL, s.c.). PolyFlex (G.C. Hanford Manufacturing Co., Syracuse, NY, USA), a general antibiotic, was administered (100 mg kg⁻¹, s.c., twice daily) during the first 3 days of recovery. The rats were housed in polycarbonate cages (10.25 inches × 18.75 inches × 8 inches) individually and the room was maintained at 26 ± 1 °C, with 40% humidity and a 12 : 12-h light–dark cycle. Post-surgical care involved manual expression of the bladder three times daily for the first 3 weeks and twice daily thereafter. On a daily basis, cage bedding was changed to prevent skin infections, animals were assessed for health (e.g. body weight, urination, defaecation, hydration), the hindlimbs were manipulated passively once through a full range of movement to maintain joint flexibility, and reflexes in the hindlimbs were assessed (i.e. withdrawal reflex and toe spread response). During the 90-day survival period there was no response to reflex testing or toe pinching, and the hindlimbs remained completely flaccid. Rats were supplied rat chow and water ad libitum. Rats in the control group were maintained under the same conditions. All of these procedures are used routinely in our laboratory and have been described in detail previously (Roy et al. 1992). The studies were approved by the UCLA Chancellor's Animal Research Committee and followed the American Physiological Society Animal Care Guidelines.

After 90 days of SI, the rats were weighed, deeply anaesthetized with sodium pentobarbital (50 mg kg⁻¹ body weight, i.p.) and decapitated. Non-operated, age-matched control rats were killed at the same time points. The MG and TA muscles were excised bilaterally, cleaned of excess connective tissue and fat, and wet weighed. The muscles were frozen close to their in situ physiological length in melting isopentane cooled in liquid nitrogen and stored at −70 °C until further analysis.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

Cross-sections 20 µm thick from the midbelly of the MG and TA were cut in a cryostat at −20 °C. The sections were placed into precooled microcentrifuge tubes and stored at −70 °C. Myofibrillar protein was isolated and prepared as described by Talmadge & Roy (1993). The gels were scanned with an Alpha Innotech Corporation IS-1000 Digital Imaging System densitometer (San Leandro, CA, USA) to quantify the MHC isoforms.

Fibre type and size procedures

Serial cross-sections (10 µm thick) from the midbelly of the right MG and TA muscles were cut in a cryostat maintained at −20 °C and mounted onto gelatin-coated slides. The MHC profiles of ∼100 fibres from a deep (d, a region of the muscle near the bone) and superficial (s, a region of the muscle away from the bone) region of the MG and TA were determined using a series of monoclonal antibodies specific to rat MHC isoforms (see Table 1 for antibody specificity) as used previously (Grossman et al. 1998). In addition, the CSAs of all type-identified fibres were determined using the Scion image processing system (Frederick, MD, USA). Briefly, the fibre cross-sections were incubated overnight with the monoclonal antibodies at 4 °C. The avidin–biotin immunohistochemical procedure was used to localize and amplify the antigen–antibody binding complex (Vectastain ABC kits, Vector Laboratories, Burlingame, CA, USA). Additional sections without primary antibody were incubated to control for non-specific binding. Fibres were considered to contain a specific MHC if there was a visually detectable reaction in the fibre to the appropriate monoclonal antibody. The fibre type composition was deduced from a comparison of the reaction of the different antibodies, e.g. a pure type IIx fibre reacted with the D9 but not the F3 antibody and a I + IIa fibre reacted with the slow, fast, 71 and 35 antibodies.

Table 1.

Monoclonal antibody specificity for myosin heavy chain isoforms

	MHC isoform
MAb designation	I	IIa	IIx	IIb	Emb
Slow	+	–	–	–	–
Fast	–	+	+	+	–
71	–	+	–	–	–
35	+	+	–	+	–
F3	–	–	–	+	–
D9	–	–	+	+	–
Dev	–	–	–	–	+

Each monoclonal antibody (MAb) bound to specific myosin heavy chain (MHC) isoforms as described by Schiaffino et al. (1989) and by the supplier's instructions. A ‘+’ indicates a positive reaction and a ‘–’ indicates no reaction. Fast, slow and developmental (Dev) antibodies were purchased from Novocastra (Burlingame, CA, USA). Antibodies 71, 35, F3 and D9 were provided by Dr Schiaffino (Padova, Italy).

Statistical analyses

All data are presented as the mean ± SEM. One-way analyses of variance (anova) followed by Tukey post-hoc tests were used to determine statistical significance. The 0.05 level of probability was established for statistical significance.

Results

Body and muscle weights

The mean body weight of the SI group progressively decreased during the first week after surgery. Thereafter, the body weights of the SI rats increased in parallel with the control group. At 90 days post-SI, the mean body weights were ∼20% lower in the SI than age-matched control rats (control = 280 ± 8 g; SI = 220 ± 6 g). This body weight response after SI surgery is similar to that reported previously (Grossman et al. 1998). The mean absolute and relative (to body weight) MG muscle weights were ∼46 and 40% of control, respectively (Fig. 1). The TA showed less atrophy, with the mean absolute and relative weights being 55 and 72% of control, respectively.

Fig. 1.

Absolute (MWt, mg) and relative (MWt, mg g⁻¹ body weight) weights of the medial gastrocnemius (MG) and tibialis anterior (TA) muscles after 90 days of inactivity induced by spinal cord isolation (SI) expressed as a percentage of age-matched control values. Bars are SEM. Asterisk, significantly different from control at P < 0.05.

MHC composition

Both the MG and the TA muscles are mixed, fast muscles containing primarily fast MHC isoforms. The percentage composition of types I, IIa, IIx and IIb MHC isoforms in the MG and TA of control rats was ∼9 ± 1, 9 ± 1, 33 ± 1 and 49 ± 1% and ∼4 ± 1, 15 ± 1, 29 ± 1, and 52 ± 1%, respectively. Ninety days of inactivity shifted the MHC profile toward the faster isoforms, i.e. the percentage MHC composition was 0.5 ± 1, 0, 39 ± 2 and 60 ± 2% for the MG and 0, 0, 57 ± 1 and 43 ± 1% in the TA. Interestingly, SI resulted in a significant increase of type IIx (from 33 to 39%) and IIb (from 49 to 60%) in the MG and increase of type IIx (from 29 to 57%) and decrease of IIb (from 52 to 43%) in the TA (Fig. 2). These data suggest that type IIb is the default MHC isoform in the inactive MG and type IIx is the default MHC isoform in the inactive TA.

Fig. 2.

Myosin heavy chain (MHC) isoform composition as determined by gel electophoresis and expressed as a percentage of total MHC for the MG (A) and TA (B) muscles of control and SI rats. Abbreviations as in Fig. 1. Bars are SEM. Asterisk, significantly different from control at P < 0.05.

Fibre type composition

Both the MG and the TA are compartmentalized relative to fibre type composition. In control rats, the MGd region comprised 20 ± 2, 9 ± 2, 43 ± 4 and 15 ± 3% pure type I, IIa, IIx and IIb fibres, respectively, whereas the MGs region contained primarily type IIb (76 ± 6%) and IIa + IIx (14 ± 5%) fibres (Fig. 3). Long-term inactivity resulted in a slow to fast shift in both regions. The major changes in the MGd included the disappearance of pure type I and pure type IIa fibres, a significant increase in type IIb fibres, and a de novo appearance of type I + IIx fibres. In the MGs, 97 ± 2% of the fibres were type IIb after 90 days of SI.

Fig. 3.

Fibre type composition as determined by immunohistochemistry and expressed as a percentage of the total number of fibres sampled in the deep (close to the bone) and superficial (away from the bone) regions of the MG of control (A) and SI (B) rats. Percentage change from control (C) for each fibre type in SI rats. Abbreviations as in Figs 1 and 2. Bars are SEM. Asterisk, significantly different from control at P < 0.05.

In control rats, the TAd region comprised 17 ± 2, 25 ± 2, 30 ± 2 and 21 ± 4% pure type I, IIa, IIx and IIb fibres, respectively, whereas the TAs region contained primarily type IIb (82 ± 4%) and IIa + IIx (11 ± 3%) fibres (Fig. 4). Following 90 days of inactivity, the percentage of IIx fibres dramatically increased in both regions (from 30 ± 2 to 66 ± 2% in TAd and from 5 ± 3 to 26 ± 4% in TAs), whereas the percentage type IIb fibres was unchanged in the deep region and decreased by ∼10% in the superficial region.

Fig. 4.

Fibre type composition as determined by immunohistochemistry and expressed as a percentage of the total number of fibres sampled in the deep (close to the bone) and superficial (away from the bone) regions of the TA of control (A) and SI (B) rats. Percentage change from control (C) for each fibre type in SI rats. Abbreviations as in Figs 1 and 2. Bars are SEM. Asterisk, significantly different from control at P < 0.05.

Generally, the increases in the percentages of fibres containing type IIb MHC in the MG and fibres containing type IIx MHC in the TA in SI compared with control rats are consistent with the adaptations in the MHC composition based on whole muscle gel analysis (compare Fig. 2 with Figs 3 and 4). No fibres containing embryonic or developmental MHCs were detected in either the MG or the TA of control and SI rats.

Fibre size adaptations

The percentage decrease in fibre size was greater than for muscle weight. This finding, however, is consistent with our observation of a decrease in the specific tension in the MG of SI rats (Kim et al. 2003), indicating that there is an increase in the percentage of non-contractile tissue in chronically inactive muscles.

The mean CSA of all fibre types of control rats ranged from ∼1700 to 3600 µm² in the MG (Fig. 5) and from ∼1100 to 3500 µm² in the TA (Fig. 6). In both the MG and the TA muscles the IIb fibres were the largest fibres in both the deep and the superficial regions and this status was maintained after SI. Compared with controls, there was a decrease (range ∼30–70%) in the CSA of all fibre types in both the MGd and the MGs after 90 days of SI (Fig. 5). The most common fibre type in each region showed the most atrophy, i.e. type IIx in the MGd (58% atrophy) and type IIb in the MGs (60% atrophy). The absolute amount of fibre atrophy (range ∼40–60%) was slightly less in the TA than in the MG (Fig. 6). The type IIb fibres were the largest and most common type in both regions of the TA, except that the IIx fibers were the most common in TAd, and also showed the most atrophy following SI.

Fig. 5.

Mean fibre cross-sectional areas of each fibre type from the deep and superficial regions of the MG of control (A) and SI (B) rats. Percentage change from control (C) for each fibre type in SI rats. Abbreviations as in Figs 1 and 2. Bars are SEM. Asterisk, significantly different from control at P < 0.05.

Fig. 6.

Mean fibre cross-sectional areas of each fibre type from the deep and superficial regions of the TA of control (A) and SI (B) rats. Percentage change from control (C) for each fibre type in SI rats. Abbreviations as in Figs 1 and 2. Bars are SEM. Asterisk, significantly different from control at P < 0.05.

Examples of the reaction of individual fibre types to the battery of monoclonal antibodies and of the fibre sizes for a representative portion of the deep and superficial regions of a TA from a control and an SI rat are shown in Figs 7 and 8, respectively.

Fig. 7.

Serial cross-sections (10 µm thick) of a representative superficial and deep region of a TA muscle from a control rat stained with monoclonal antibodies for specific MHC isoforms (see Table 1 for staining specificity). Scale bar = 100 µm.

Fig. 8.

Serial cross-sections (10 µm thick) of a representative superficial and deep region of a TA muscle from an SI rat stained with monoclonal antibodies for specific MHC isoforms (see Table 1 for staining specificity). Scale bar = 100 µm.

Discussion

The primary findings of the present study comparing the responses of a fast extensor (MG) and a fast flexor (TA) muscle to long-term inactivity (90 days) induced by SI are: (1) the amount of muscle and fibre atrophy is greater in the MG than TA; (2) although both muscles showed a shift toward the faster MHC isoforms, the default MHC isoform is type IIb for the MG and type IIx for the TA; (3) there is no expression of developmental or embryonic MHC isoforms in either muscle after 90 days of inactivity; and (4) the default muscle fibre size is similar in the two muscles, i.e. ∼600–1300 and 700–1500 µm² in the MG and TA, respectively. The results are consistent with the first two hypotheses, but not the third.

The response to long-term inactivity induced by SI is different for fast vs. slow muscles

Inactivity induced by SI results in a rapid (first 2 weeks) atrophy of both slow (e.g. soleus, adductor longus) and fast (e.g. MG and TA) hindlimb muscles (Grossman et al. 1998). Huey et al. (2001) reported that the slow soleus muscle atrophied by 43% in the first week after SI, and then reached a plateau (∼55% atrophy) between 15 and 60 days. From 60 to 90 days after SI, however, the soleus weight continued to decrease, i.e. to 26% of age-matched control values. There was a progressive increase in the percentage of fast MHC composition that was significant after 30 days of SI (from ∼10% type IIa to ∼10 and 15% types IIa and IIx MHC) and reached ∼35% (∼17% for both types IIa and IIx MHC) after 60 days. In addition, there was a very rapid transformation from the slow to the fast MHC isoforms between 60 and 90 days post-SI, with types IIa and IIx reaching ∼16 and 69%, respectively. There was also a small amount of type IIb detected in the soleus only after 90 days of SI.

Would similar dramatic changes in muscle weight and MHC composition observed in the slow soleus from 60 to 90 days post-SI occur in predominantly fast muscles? Roy et al. (2005) reported that the muscle weight to body weight ratios for the MG and TA were decreased by 36 and 21%, respectively, at 60 days post-SI. These ratios were 41 and 28% after 90 days of SI in the present study. Therefore, the rate of atrophy from 60 to 90 days of inactivity was slower in fast than in slow muscles. Roy et al. (2000) also reported that after 60 days of SI the MHC composition of the MG was ∼2% type I, ∼2% type IIa, ∼35% type IIx and ∼60% type IIb MHC. In the present study, we show that after 90 days of SI the MG contains almost no type I or IIa MHC, but that the percentage of type IIx (39%) and IIb (60%) remain at about the same level as found after 60 days of SI. Thus, both the rates of atrophy and of MHC transformation are lower in fast than in slow muscles from 60 to 90 days of inactivity. Whether this muscle type-specific response continues with longer periods of inactivity induced by SI is unknown.

Activity-independent effects on muscle fibre size

The SI model eliminates all supraspinal, infraspinal and peripheral afferent input, i.e. all neural activity-dependent influences, on the motoneurons located in the isolated region of the spinal cord, while leaving the motoneuron–muscle connectivity intact (Grossman et al. 1998). In effect, the activity-independent neurotrophic influences are maintained following SI surgery. By contrast, denervation eliminates both activity-dependent and -independent influences. A comparison of these two models therefore should provide insight into the role of activity-independent neural influences on muscle mass. Recently, Hyatt et al. (2003) reported that compared with control values the mean relative (muscle weight to body weight) MG and TA weights were, respectively, ∼64 and 71% in SI rats and 46 and 48% in denervated rats 2 weeks after surgery. This differential muscle atrophy was even more disparate by 28 days, i.e. ∼60 and ∼25% for the mean absolute and relative weights for both muscles in SI and denervated rats, respectively.

At the single fibre level, Borisov et al. (2001) reported that the mean CSA of the fast fibres in the TA of young adult rats decreased by 45, 69 and 88% from control values after 1, 2 and 4 months of denervation, respectively. Similar results were observed for another primary fast flexor muscle, the extensor digitorum longus (EDL) (Carlson et al. 2002). By contrast, the mean CSA of all fibres containing some fast MHC isoforms in the TA was between 33 and 46% smaller after 2 months of SI (Roy et al. 2005) and 40–60% smaller after 3 months of SI (present study). Thus, it appears that the neural activity-independent influences remaining in the SI model have a strong effect in maintaining both muscle and fibre size during prolonged periods of inactivity.

In general, the adaptations in fibre size were similar in the deep and superficial regions of each muscle. Furthermore, all muscle fibres atrophied such that the mean fibre CSA in SI rats was relatively similar in both regions of both muscles. In the MG, the two most common types of fibres identified in both regions (type IIx and type IIb) showed a similar amount of atrophy, i.e. ∼60%. In the TA, type IIx and type IIb fibres also were the most common fibre types. The type IIx fibres atrophied similarly in the two regions (∼40%), whereas the percentage atrophy of type IIb fibres was somewhat higher in the superficial (60%) than in the deep (40%) region. These data indicate that activity-independent influences account for ∼50% of muscle fibre size in SI rat MG and TA muscles and are not region-specific, particularly for the MG.

Inactivity induced by SI results in muscle-specific MHC default modes and no appearance of developmental MHC isoforms

The present results indicate that the effects of inactivity produced by SI on the MHC composition were muscle-specific but not region-specific. In effect, the default MHC mode for the MG was type IIb and that for the TA was IIx. In general, the shifts of fibre type composition were similar in the deep and superficial region in each muscle. These data confirm and extend the results from a previous study based on the MHC profile of a sample of single fibres mechanically isolated from the MG and TA for the determination of myonuclear domains (Roy et al. 2005). Both changes in the fibre type and the fibre size contributed to the shift in MHC composition after SI. There was a significant increase in the percentage of pure type IIb fibres in both regions of the MG and the percentage atrophy of the type IIb fibres was similar to that of the other major fibre type, i.e. pure type IIx fibres. Similarly, there was a significant increase in the percentage of pure type IIx fibres in both regions of the TA and the percentage atrophy of the type IIx fibres was similar to that of the other major fibre type, i.e. pure IIb fibres.

The adaptations in the phenotype composition of fast flexors are in the same direction after either denervation or TTX treatment, but are different for the fast extensors. Two weeks of denervation of either the EDL (Michel et al. 1996; Jakubiec-Puka et al. 1999) or the gastrocnemius (Jakubiec-Puka et al. 1999) resulted in a general decrease in type IIb and increases in types IIa and IIx MHCs. Similarly, 3 weeks of denervation of EDL (Jakubiec-Puka et al. 1990) and 4 weeks of denervation of the plantaris, a fast agonist of the MG (Haddad et al. 1997), resulted in a decrease in type IIb and increase in type IIa MHC. Windisch et al. (1998) reported that after denervation of the EDL, the percentage of IIb fibres slowly declined from ∼50 to < 10% over a period of 2 months, whereas no pure type IIx fibres were observed and the percentage of type IIa fibres increased from ∼25 to 90%. After 2 weeks of TTX treatment, the percentage of fibres in the EDL containing type IIb myosin decreased, with a general increase in types IIa and IIx (Michel et al. 1996). The percentage of fibres containing some type IIx was significantly higher in both the deep and the superficial regions of the MG after 2 or 4 weeks of TTX treatment (Cormery et al. 2000). Combined these data suggest that the phenotypic adaptations in fast flexors are modulated similarly under a variety of inactivity models, i.e. a default mode of type IIx (and IIa) MHC. In contrast in the fast extensors, SI results in a shift to the fastest MHC isoforms, whereas the default mode in the denervated and TTX-treated muscles is type IIa and IIx MHC. The reasons for this differential response in the extensor muscles are unknown.

The presence of fibres containing developmental (neonatal and/or embryonic) MHC is interpreted routinely as a sign of denervation and/or re-innervation (Schiaffino et al. 1988). Muscles from adult control rats normally do not express these MHC isoforms. As early as 2 weeks after denervation, ∼30% of the fibres in the EDL contained some embryonic MHC (none in control) (Michel et al. 1996) and ∼75% of the fibres in both the deep and the superficial regions of the MG contained some developmental (embryonic and/or neonatal) MHC after 4 weeks (Cormery et al. 2000). Similar results were observed in the EDL (Michel et al. 1996), TA (Schiaffino et al. 1988) and MG (Cormery et al. 2000) after TTX application on a peripheral nerve, an agent that prevents action potentials from reaching the affected muscles by blocking the Na⁺ channels. Furthermore, 4 weeks of either denervation or TTX treatment resulted in ∼90% of the fibres in the MG expressing some developmental MHC (Cormery et al. 2000). Thus, both fast flexor and fast extensor muscles expressed developmental MHCs following denervation or TTX treatment. By contrast, there were no fibres in either the rat MG or the TA that contained any developmental MHCs after 90 days of SI (present study). An earlier study reported that there was no developmental MHC in the fibres of the MG after 4, 15, 30 or 60 days of SI (Roy et al. 2000). Combined, these data demonstrate the degree to which neuromuscular connectivity and activity influence the adult MHC profile in fast skeletal muscles. The mechanism for the expression of developmental MHCs in the TTX-treated, but not the SI, rats is unknown. One possibility is the different effects of these interventions on the motoneurons innervating the affected muscles, i.e. the motoneurons are activated normally in the TTX-treated rats, whereas they are nearly silent in the SI rats.

Perspective

The present and previous (Grossman et al. 1998; Roy et al. 2000, 2005) results indicate that isolation of the motoneurons in the lumbar region of the spinal cord from supraspinal, infraspinal and peripheral input results in near inactivity (Zhong et al. 2002) and a progressive decrease in fibre size and adaptations in fibre phenotype in the associated hindlimb muscles. The uniqueness of this inactivity model is that the motoneuron–muscle connectivity is intact, thus maintaining any neural activity-independent (i.e. neurotrophic) influences on the muscles. The combined results indicate that these influences are muscle-specific, i.e. slow vs. fast and/or extensor vs. flexor. In addition, because SI produces near zero activity in the hindlimb muscles the effects of a known amount and pattern of neuromuscular activity can be imposed on the muscles via the intact nerve to determine efficacious stimulation countermeasures of activity-induced atrophy and phenotypic adaptations.