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Myofiber atrophy is the final outcome of muscle wasting induced by catabolic factors such as glucocorticoids and thyroid hormones. We set up an in vitro system to define the catabolic reaction based on myotube atrophy. Both mouse [C.sub.2][C.sub.12] and rat L6 cells were used. [C.sub.2][C.sub.12] myotube formation was improved by replacing horse serum with the serum substitute Ultroser G. A new method was developed to quantify size changes of large (0.5-1 mm) myotubes only, excluding remaining myoblasts and small myotubes. Dexamethasone reduced myotube size by 30% in L6 but not in [C.sub.2][C.sub.12] myotubes. Expression of the glucocorticoid receptor was twofold higher in L6 myotubes than in [C.sub.2][C.sub.12] myotubes. In both cell lines, 3,3',5-triiodo-L-thyronine ([T.sub.3]) did not induce a significant size reduction. Expression of the major [T.sub.3] receptor ([T.sub.3]R[beta]1) was higher in L6 myotubes. We investigated whether the changes in myotube size are related to changes in atrogin-1 expression, as this enzyme is thought to be a key factor in the initiation of muscle atrophy. Dexamethasone induced a twofold increase of atrogin-1 mRNA; again, only L6 myotubes were susceptible. Interestingly, atrogin-1 expression in Ultroser G-fused [C.sub.2][C.sub.12] myotubes was lower than that in horse serum-fused myotubes. Furthermore, dexamethasone treatment increased atrogin-1 expression only in horse serum-fused myotubes but not in Ultroser G-fused myotubes. Ultroser G-induced fusion may result in atrophy-resistant [C.sub.2][C.sub.12] myotubes. Therefore, [C.sub.2][C.sub.12] myotubes offer an ideal system in which to study skeletal muscle atrophy because, depending on differentiation conditions, [C.sub.2][C.sub.12] cells produce atrophy-inducible and atrophy-resistant myotubes. glucocorticoids; nuclear receptors; atrogin

We examined satellite cell content and the activity of satellite cell progeny in tibialis anterior muscles of young (15 weeks) and aging (101 weeks) Brown Norway (BN) rats, after they were exposed for 50 days to a standardized and highly reproducible regime of chronic low-frequency electrical stimulation. Chronic low-frequency electrical stimulation was successful in inducing fast-to-slow fiber-type transformation, characterized by a 2.3-fold increase in the proportion of IIA fibers and fourfold and sevenfold decreases in the proportion of IID/X and IIB fibers in both young and aging BN rats. These changes were accompanied by a twofold increase in the satellite cell content in both the young and aging groups; satellite cell content reached a level that was significantly higher in the young group (p < .04). The total muscle precursor ceil content (i.e., satellite cells plus progeny), however, did not differ between groups, because there was a greater number of satellite cell progeny passing through the proliferative and differentiative compartments of the aging group. The resulting 1.5-fold increase in myonuclear content was similar in the young and aging groups. We conclude that satellite cells and satellite cell progeny of aging BN rats possess an unaltered capacity to contribute to the adaptive response.

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.

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

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

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

Myofiber atrophy is the final outcome of muscle wasting induced by catabolic factors such as glucocorticoids and thyroid hormones. We set up an in vitro system to define the catabolic reaction based on myotube atrophy. Both mouse C2C12 and rat L6 cells were used. C2C12 myotube formation was improved by replacing horse serum with the serum substitute Ultroser G. A new method was developed to quantify size changes of large (0.5–1 mm) myotubes only, excluding remaining myoblasts and small myotubes. Dexamethasone reduced myotube size by 30% in L6 but not in C2C12 myotubes. Expression of the glucocorticoid receptor was twofold higher in L6 myotubes than in C2C12 myotubes. In both cell lines, 3,3′,5-triiodo-L-thyronine (T3) did not induce a significant size reduction. Expression of the major T3 receptor (T3Rβ1) was higher in L6 myotubes. We investigated whether the changes in myotube size are related to changes in atrogin-1 expression, as this enzyme is thought to be a key factor in the initiation of muscle atrophy. Dexamethasone induced a twofold increase of atrogin-1 mRNA; again, only L6 myotubes were susceptible. Interestingly, atrogin-1 expression in Ultroser G-fused C2C12 myotubes was lower than that in horse serum-fused myotubes. Furthermore, dexamethasone treatment increased atrogin-1 expression only in horse serum-fused myotubes but not in Ultroser G-fused myotubes. Ultroser G-induced fusion may result in atrophy-resistant C2C12 myotubes. Therefore, C2C12 myotubes offer an ideal system in which to study skeletal muscle atrophy because, depending on differentiation conditions, C2C12 cells produce atrophy-inducible and atrophy-resistant myotubes. [ABSTRACT FROM AUTHOR]

Examines satellite cell content and the activity of satellite cell progeny in tibialis anterior muscles of young and aging Brown Norway rats. Changes in the skeletal muscle fibers associated with aging; Assessment of fiber type under study; Examination of damaged fibers.

The relationship between heat shock protein (Hsp) expression and meat quality was assessed in pigs. Carcasses from 2 h and 6 h transported pigs had higher temperatures and lower pH and water holding capacity values in the longissimus dorsi and gluteus maximus superficialis muscles. Long journeys were associated with increased creatine kinase (CK) levels. Higher CK levels are indicative of physical stress, as the enzyme is released from muscle fibers as a result of intense muscular exertion. These physiological and enzymatic changes were correlated with increased Hsp70 and decreased Hsp90 expression levels in both skeletal muscles. Animals whose cells contained high levels of Hsp may have had an advantage due to the protective role conferred by Hsp. Reduced Hsp levels were indicative of a higher meat quality and a good welfare of the transported pigs. The stress response declined over time in response to the same stress, such as a 6 h transport stress. [ABSTRACT FROM AUTHOR]

The objective of the present study was to investigate the role of heat shock proteins (Hsps) as potential stress response marker in several organs of transported pigs. Constitutive (Hsp90, Hsp70 and Hsp27) and inducible (Hsp72 and Hsp86) Hsps expressed in skeletal muscle, heart, liver and kidney of transported young pigs were investigated. The study comprised 13 German Landrace line pigs (mean weight, approx. 35 +/- 1 kg). Eight animals (n = 8) were transported for a period of 6 h, whereas the control group (n = 5) was kept under normal housing conditions. Identification of Hsps was performed by immunoblot using porcine specific antibodies. Although the 5 Hsps were regularly detected in all porcine tissues, no clear up-regulation could be observed due to transportation. Densitometrical analysis of the immunoblots revealed an unexpected result. Every tissue had a significant reduction of at least 2 Hsp members: Kidney (Hsp90 and Hsp70), M. longissimus dorsi (Hsp90 and Hsp72) and M. gluteus maximus superficialis (Hsp90 and Hsp86), liver (Hsp90, Hsp86 and Hsp27). The heart was most affected, all Hsps were significantly reduced by 26% to 41% after 6 h of transportation. The regular reduction of the large Hsp90 and Hsp86 in nearly all tissues examined point to a new and critical role these Hsps might have in counteracting short-term stress reactions. The fast reduction of Hsps in fatal organs such as heart and kidney may have a relation to organ failure. Hsp90 level may therefore serve as a potential marker for the stress pigs are suffering during transportation.