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5'AMP-activated protein kinase (AMPK) signaling initiates adaptive changes in skeletal muscle fibers that restore homeostatic energy balance. The purpose of this investigation was to examine, in rats, the fiber-type protein expression patterns of the [alpha]-catalytic subunit isoforms in various skeletal muscles, and changes in their respective contents within the tibialis anterior (TA) after chronic low-frequency electrical stimulation (CLFS; 10 Hz, 10 h daily), applied for 4 [+ or -] 1.2 or 25 [+ or -] 4.8 days. Immunocytochemical staining of soleus (SOL) and medial gastrocnemius (MG) showed that 86 [+ or -] 4.1 to 97 [+ or -] 1.4% of type IIA fibers stained for both the [alpha]1- and [alpha]2-isoforms progressively decreased to 63 [+ or -] 12.2% of type IID/X and 9 [+ or -] 2.4% of IIB fibers. 39 [+ or -] 11.4% of IID/X and 83 [+ or -] 7.9% of IIB fibers expressed only the [alpha]2 isoform in the MG, much of which was localized within nuclei, [alpha]1 and [alpha]2 contents, assessed by immunoblot, were lowest in the white gastrocnemius [WG; 80% myosin heavy chain (MHC) IIb; 20% MHCIId/x]. Compared with the WG, [alpha]1 content was 1.6 [+ or -] 0.08 (P < 0.001) and 1.8 [+ or -] 0.04 (P < 0.0001)-fold greater in the red gastrocnemius (RG: 13%, MHCIIa) and SOL (21%, MHCIIa), respectively, and increased in proportion to MHCIIa content. Similarly, [alpha]2 content was 1.4 [+ or -] 0.10 (P < 0.02) and 1.5 [+ or -] 0.07 (P < 0.001)-fold greater in RG and SOL compared with WG. CLFS induced 1.43 [+ or -] 0.13 (P < 0.007) and 1.33 [+ or -] 0.08 (P < 0.009)-fold increases in the [alpha]1 and [alpha]2 contents of the TA and coincided with the transition of faster type IIB and IID/X fibers toward IIA fibers. These findings indicate that fiber types differ with regard to their capacity for AMPK signaling and that this potential is increased by CLFS. myosin heavy chain; fast-twitch muscle; slow-twitch muscle

The purpose of this investigation was to examine the temporal changes in uncoupling protein (UCP)-3 expression, as well as related adaptive changes in miitochondrial density and fast-to-slow fiber type transitions during chronically enhanced contractile activity. We examined the effects of 1-42 days of chronic low-frequency electrical stimulation (CLFS), applied to rat tibialis anterior (TA) for l0 h/day, on the expression of UCP-3 and concomitant changes in myosin heavy chain (MHC) protein expression and increases in oxidative capacity. UCP-3 protein content increased from 1 to 12 days, reaching 1.5-fold over control (P < 0.0005); it remained elevated for up to 42 days. In contrast, UCP-3 mRNA decreased in response to CLFS, reaching a level that was threefold lower than control (P < 0.0007). The activities of the mitochondrial reference enzymes citrate synthase (EC 4.1.3.7) and 3-hydroxyacyl-CoA-dehydrogenase (EC 1.1.1.35), which are known to increase in proportion to mitochondrial density, progressively increased up to an average of 2.3-fold (P < 0.00001). These changes were accompanied by fast-to-slow fiber type transitions, characterized by a shift in the pattern of MHC expression (P mitochondrial density; fiber type transitions

This study focuses on the effects of mechanical unloading of rat soleus muscle on the isoform patterns of the three troponin (Tn) subunits: troponin T (TNT), troponin I (TnI), and troponin C (TnC). Mechanical unloading was achieved by hindlimb unloading (HU) for time periods of 7, 15, and 28 days. Relative concentrations of slow and fast TnT, TnI, and TnC isoforms were assessed by electrophoretic and immunoblot analyses. HU induced profound slow-to-fast isoform transitions of all Tn subunits, although to different extents and with different time courses. The effectiveness of the isoform transitions was higher for TnT than for TnI and TnC. Indeed, TnI and TnC encompassed minor partial exchanges of slow isoforms with their fast counterparts, whereas the expression pattern of fast TnT isoforms (TnTf) was largely increased after HU. Moreover, slow and fast isoforms of the different Tn were not affected in the same manner by HU. This suggests that the slow and fast counterparts of the Tn subunit isoforms are regulated independently in response to HU. The changes in TnTf composition occurred in parallel with previously demonstrated transitions within the pattern of the fast myosin heavy chains in the same muscles. hindlimb suspension; isoform transition; troponin T, I, and C.

A study was conducted to identify early changes in gene expression during the fast-to-slow transition induced by chronic low-frequency stimulation. Total RNA was extracted from 12-h-stimulated tibialis anterior muscles of rats. A cDNA about 300 bp in length was identified as corresponding to the mRNA of the muscle LIM protein (MLP). Results suggested that the MLP has a critical role during the rearrangement of cytoskeletal and/or myofibrillar structures in transforming muscle fibers.

The absolute amounts of messenger RNA for four isoforms of the rat myosin heavy chain (MHC) were measured using a quantitative reverse transcriptase-polymerase chain reaction to determine the effect of chronic low-frequency stimulation on the proportion of the MHC isoforms. Results reveal that the fast twitch muscle of the rat extensor digitorum longus has a limited capacity for fast-to-slow conversion.

Research shows that rabbit fast-twitch muscle fibres exhibit modified isometric contractile properties and electromyographic activity when they have been stimulated with chronic low-frequency signals (CLFS). Modified levels of protein in the sarcoplasmic reticulum sodium potassium adenosinetriphosphatase were observed when tibialis anterior muscles were exposed to ten hertz signals for a duration of ten minutes. Reduced levels of ryanodine receptor, dihydropyridine receptor and triadin concentrations were also observed after 20 days CLFS exposure.

Nuclear magnetic resonance spectroscopy, electron microscopy, histochemical fiber typing and calcium measurements in cultured skeletal muscle cells obtained from genetically engineered mice were performed to study the creatinine kinase (CK)-catalyzed reactions in the skeletal muscle network system. The results indicated that the CK system regulates both Ca(super 2+) accumulation and release in skeletal muscle fibers.

Deficiency in parvalbumin increases fatigue resistance in fast-twitch muscle and upregulates mitochondria. Am J Physiol Cell Physiol 281: C114-C122, 2001.--The soluble [Ca.sup.2+]-binding protein parvalbumin (PV) is expressed at high levels in fast-twitch muscles of mice. Deficiency of PV in knockout mice (PV -/-) slows down the speed of twitch relaxation, while maximum force generated during tetanic contraction is unaltered. We observed that PV-deficient fast-twitch muscles were significantly more resistant to fatigue than were the wild type. Thus components involved in [Ca.sup.2+] homeostasis during the contraction-relaxation cycle were analyzed. No upregulation of another cytosolic [Ca.sup.2+]-binding protein was found. Mitochondria are thought to play a physiological role during muscle relaxation and were thus analyzed. The fractional volume of mitochondria in the fast-twitch muscle extensor digitorum longus (EDL) was almost doubled in PV -/- mice, and this was reflected in an increase of cytochrome c oxidase. A faster removal of intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) 200-700 ms after fast-twitch muscle stimulation observed in PV -/- muscles supports the role for mitochondria in late [[[Ca.sup.2+]].sub.i] removal. The present results also show a significant increase of the density of capillaries in EDL muscles of PV -/- mice. Thus alterations in the dynamics of [Ca.sup.2+] transients detected in fast-twitch muscles of PV -/- mice might be linked to the increase in mitochondria volume and capillary density, which contribute to the greater fatigue resistance of these muscles. muscle fatigue; calcium-binding protein; EF hand; compensation

Muscle LIM protein is upregulated in fast skeletal muscle during transition toward slower phenotypes. Am J Physiol Cell Physiol 280: C273-C279, 2001.--Muscle LIM protein (MLP) is constitutively expressed in slow, but undetectable in fast, muscles of the rat. Here we show that MLP was upregulated at both the mRNA and protein levels under experimental conditions leading to transitions from fast to slower phenotypes. Chronic low-frequency stimulation and mechanical overloading by synergist removal both induced fast-to-slow shifts in myosin heavy chain (MHC) isoforms and expression of MLP in fast muscles. High amounts of MLP mRNA and protein were also present in fast muscles of the myotonic, hyperactive ADR mouse. Hypothyroidism evoked shifts in myosin composition toward slower isoforms and increased the MLP protein content of soleus (SOL) muscle but failed to induce MLP in fast muscles. Unweighting by hindlimb suspension elicited slow-to-fast transitions in MHC expression without altering MLP levels in SOL muscle. Hyperthyroidism shifted the MHC pattern toward faster isoforms but did not affect MLP content in SOL muscle. We conclude that alterations in MLP expression are associated with transitions from fast to slower phenotypes but not with slow-to-fast muscle fiber transitions. ADR mouse; chronic low-frequency stimulation; fiber type transition; hindlimb suspension; mechanical overloading; myotonia; thyroid hormone

Fiber type-specific expression of major proteolytic systems in fast- to slow-transforming rabbit muscle. Am J Physiol Cell Physiol 280: C239-C247, 2001.--The present study investigates the role of two major proteolytic systems in transforming rabbit and rat muscles. The fast-to-slow transformation of rabbit muscle by chronic low-frequency stimulation (CLFS) induces fast-to-slow transitions of intact, mature fibers and replacement of degenerating fibers by newly formed slow fibers. Ubiquitination, an indicator of the ATP-dependent proteasome system, and calpain activity were measured in homogenates of control and stimulated extensor digitorum longus muscles. Calpain activity increased similarly (~2-fold) in stimulated rat and rabbit muscles. CLFS had no effect on protein ubiquitination in rat muscle but led to elevations in ubiquitin protein conjugates in rabbit muscle. Immunohistochemistry was used to study the distribution of [micro]-calpain and m-calpain and of ubiquitinated proteins in myosin heavy chain-based fiber types. The findings suggest that both proteolytic systems are involved in fiber transformation and replacement. Transforming mature fibers displayed increases in [micro]-calpain and accumulation of ubiquitin protein conjugates. The majority of these fibers were identified as type IIA. Enhanced ubiquitination was also observed in degenerating and necrotic fibers. Such fibers additionally displayed elevated m-calpain levels. Conversely, p94, the skeletal muscle-specific calpain, decayed rapidly after stimulation onset and was hardly detectable after 4 days of CLFS. calpains; chronic low-frequency stimulation; fiber type; ubiquitination

Stevens, Laurence, Carole Firinga, Barbel Gohlsch, Bruno Bastide, Yvonne Mounier, and Dirk Pette. Effects of unweighting and clenbuterol on myosin light and heavy chains in fast and slow muscles of rat. Am J Physiol Cell Physiol 279: C1558-C1563, 2000.--To investigate, the plasticity of slow and fast muscles undergoing slow-to-fast transition, rat soleus (SOL), gastrocnemius (GAS), and extensor digitorum longus (EDL) muscles were exposed for 14 days to 1) unweighting by hindlimb suspension (HU), or 2) treatment with the [Beta.sub.2]-adrenergic agonist clenbuterol (CB), or 3) a combination of both (HU-CB). In general, HU elicited atrophy, CB induced hypertrophy, and HU-CB partially counteracted the HU-induced atrophy. Analyses of myosin heavy (MHC) and light chain (MLC) isoforms revealed HU- and CB-induced slow-to-fast transitions in SOL (increases of MHCIIa with small amounts of MHCIId and MHCIIb) and the upregulation of the slow MHCIa isoform. The HU- and CB-induced changes in GAS consisted of increases in MHCIId and MHCIIb ('fast-to-faster transitions'). Changes in the MLC composition of SOL and GAS consisted of slow-to-fast transitions and mainly encompassed an exchange of MLC1s with MLC1f. In addition, MLC3f was elevated whenever MHCIId and MHCIIb isoforms were increased. Because the EDL is predominantly composed of type IID and IIB fibers, HU, CB, and HU-CB had no significant effect on the MHC and MLC patterns. extensor digitorum longus; gastrocnemius; hindlimb suspension; myosin; slow-to-fast transition; soleus

Satellite cell proliferation in low frequency-stimulated fast muscle of hypothyroid rat. Am J Physiol Cell Physiol 279: C682-C690, 2000.--Satellite cell proliferation was assessed in low-frequency-stimulated hypothyroid rat fast-twitch muscle by 5-bromo-2'-deoxyuridine (BrdU) labeling and subsequent staining of labeled muscle nuclei, and by staining for proliferating cell nuclear antigen (PCNA). BrdU labeling and PCNA staining were highly correlated and increased approximately fourfold at 5 days of stimulation, decayed thereafter, but remained elevated over control in 10-and 20-day stimulated muscles. Myogenin mRNA was ~4-fold elevated at 5 days and 1.5-fold at 10 days. Staining for myogenin protein yielded results similar to that for PCNA and BrdU. Furthermore, a detailed examination of the pattern of myogenin staining revealed that the number of myogenin-positive nuclei was elevated in the fast pure IIB fiber population at 5 and 10 days of chronic low-frequency stimulation. By 20 days, myogenin staining was observed in transforming fast fibers that coexpressed embryonic and adult myosin heavy chain isoforms. In the slower fiber populations (i.e., IIA and I), myogenin-positive transforming fibers that coexpressed embryonic myosin heavy chain, appeared already at 5 days. Thus the satellite cell progeny on slower fibers seemed to proliferate less and to fuse earlier to their associated fibers than the satellite cell progeny on fast fibers. We suggest that the increase in muscle nuclei of the fast fibers might be a prerequisite for fast-to-slow fiber type transitions. chronic low-frequency stimulation; muscle fiber transformation; myogenin; myosin heavy chain isoforms

Calpain activity in fast, slow, transforming, and regenerating skeletal muscles of rat. Am J Physiol Cell Physiol 279: C639-C647, 2000.--Fiber-type transitions in adult skeletal muscle induced by chronic low-frequency stimulation (CLFS) encompass coordinated exchanges of myofibrillar protein isoforms. CLFS-induced elevations in cytosolic [Ca.sup.2+] could activate proteases, especially calpains, the major [Ca.sup.2+]-regulated cytosolic proteases. Calpain activity determined by a fluorogenic substrate in the presence of unaltered endogenous calpastatin activities increased twofold in low-frequency. stimulated extensor digitorum longus (EDL) muscle, reaching a level intermediate between normal fast- and slow-twitch muscles. [micro] and m-calpains were delineated by a calpain-specific zymographical assay that assessed total activities independent of calpastatin and distinguished between native and processed calpains. Contrary to normal EDL, structure-bound, namely myofibrillar and microsomal calpains, were abundant in soleus muscle. However, the fast-to-slow conversion of EDL was accompanied by an early translocation of cytosolic [micro]-calpain, suggesting that myofibrillar and microsomal [micro]-calpain was responsible for the twofold increase in activity and thus involved in controlled proteolysis during fiber transformation. This is in contrast to muscle regeneration where m-calpain translocation predominated. Taken together, we suggest that translocation is an important step in the control of calpain activity in skeletal muscle in vivo. calpain activation; calpastatin; chronic low-frequency stimulation; fast-to-slow transition; translocation

Carroll, Stefanie, Pierluigi Nicotera, and Dirk Pette. Calcium transients in single fibers of low-frequency stimulated fast-twitch muscle of rat. Am. J. Physiol. 277 (Cell Physiol. 46): C1122-C1129, 1999.--[Ca.sup.2+] transients were investigated in single fibers isolated from rat extensor digitorum longus muscles exposed to chronic low-frequency stimulation for different time periods up to 10 days. Approximately 2.5-fold increases in resting [Ca.sup.2+] concentration ([[Ca.sup.2+]]) were observed 2 h after stimulation onset and persisted throughout the stimulation period. The elevated [[Ca.sup.2+]] levels were in the range characteristic of slow-twitch fibers from soleus muscle. In addition, we noticed a transitory elevation of the integral [[Ca.sup.2+]] per pulse with a maximum (~5-fold) after 1 day. Steep decreases in rate constant of [[Ca.sup.2+]] decay could be explained by an immediate impairment of [Ca.sup.2+] uptake and, with longer stimulation periods, by an additional loss of cytosolic [Ca.sup.2+] binding capacity resulting from a decay in parvalbumin content. A partial recovery of the rate constant of [[Ca.sup.2+]] decay in 10-day stimulated muscle could be explained by an increasing mitochondrial contribution to [Ca.sup.2+] sequestration. calcium buffering; free calcium; mitochondria; parvalbumin; sarcoplasmic reticulum calcium-adenosinetriphosphatase

Stevens, Laurence, Karim R. Sultan, Heidemarie Peuker, Barbel Gohlsch, Yvonne Mounier, and Dirk Pette. Time-dependent changes in myosin heavy chain mRNA and protein isoforms in unloaded soleus muscle of rat. Am. J. Physiol. 277 (Cell Physiol. 46): C1044-C1049, 1999.--Time-dependent changes in myosin heavy chain (MHC) isoform expression were investigated in rat soleus muscle unloaded by hindlimb suspension. Changes at the mRNA level were measured by RT-PCR and correlated with changes in the pattern of MHC protein isoforms. Protein analyses of whole muscle revealed that MHCI decreased after 7 days, when MHCIIa had increased, reaching a transient maximum by 15 days. Longer periods led to inductions and progressive increases of MHCIId(x) and MHCIIb. mRNA analyses of whole muscle showed that MHCIId(x) displayed the steepest increase after 4 days and continued to rise until 28 days, the longest time period investigated. MHCIIb mRNA followed a similar time course, although at lower levels. MHCI[Alpha] mRNA, present at extremely low levels in control soleus, peaked after 4 days, stayed elevated until 15 days, and then decayed. Immunohistochemistry of 15-day unloaded muscles revealed that MHCI[Alpha] was present in muscle spindles but at low amounts also in extrafusal fibers. The slow-to-fast transitions thus seem to proceed in the order MHCI[Beta] [right arrow] MHCIIa [right arrow] MHCIId(x) [right arrow] MHCIIb. Our findings indicate that MHCI[Alpha] is transiently upregulated in some fibers as an intermediate step during the transition from MHCI[Beta] to MHCIIa. hindlimb suspension; messenger ribonucleic acid; myosin heavy chain isoforms; reverse transcriptase-polymerase chain reaction; slow-to-fast transition