BDNF rescues myosin heavy chain IIB muscle fibers after neonatal nerve injury

Neonatal sciatic nerve injury is known to result in an extensive loss of lumbar motor neurons as well as the disappearance of their respective muscle fibers in the hindlimb musculature. The loss of motor neurons and muscle fibers can be prevented by immediate administration of target-derived neurotrophic factors to the site of injury. In the present study, we investigated the role of ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) in the survival and maturation of a subset of motor neurons innervating the extensor digitorum longus (EDL) and tibialis anterior (TA) muscles. We have shown that combined administration of CNTF and BDNF prevented the loss of motor units after neonatal nerve injury and contributed to the maintenance of muscle mass. Importantly, this combined neurotrophin regimen also prevented the disappearance of muscle fibers that express myosin heavy chain IIB (MyHC IIB) in both EDL and TA muscles 3 mo after neonatal sciatic nerve crush. In parallel studies, we observed a higher level of BDNF in EDL muscle during the critical period of development when motor neurons are highly susceptible to target removal. Given our previous findings that combined administration of CNTF with neurotrophin-3 (NT-3) or neurotrophin-4/5 (NT-4/5) did not result in the rescue of MyHC IIB fibers in EDL, the present results show the importance of muscle-derived BDNF in the survival and maturation of a subpopulation of motor neurons and of MyHC IIB muscle fibers during neonatal development of the neuromuscular system. motor neurons; neuromuscular development; neurotrophins     

Structural and functional responses to prolonged hindlimb suspension in rat muscle

The purpose of this study was to investigate alterations in structural and functional properties in the soleus (SOL) and extensor digitorum longus (EDL) muscles of rats after 1, 2, and 5 wk of tail suspension. Maximal O2 uptake was 19% lower after 5 wk suspension. Loss of muscle mass was greater in SOL (63%) than in EDL (22%) muscle. A reduction of type I distribution was accompanied by an increase of intermediate fiber subgroups (int I in SOL, int II in EDL). The cross-sectional area of all three fiber types was reduced by hypokinesia. The decrease in capillaries per fiber in SOL was greater than the decrease in citrate synthase and 3-hydroxyacyl-CoA dehydrogenase activities after 5 wk. No alteration in lactate dehydrogenase activity was noted. In EDL, no changes in fiber area, capillarization, and enzymatic activities occurred. Energy charge remained unchanged (0.91) whatever the muscle. These results suggest that type I fibers showed an earlier and greater susceptibility than type II fibers to suspension which is also accompanied by a decreased aerobic capacity.     

Absolute amounts and diffusibility of HSP72, HSP25, and αB-crystallin in fast- and slow-twitch skeletal muscle fibers of rat

Heat shock proteins (HSPs) are essential for normal cellular stress responses. Absolute amounts of HSP72, HSP25, and αB-crystallin in rat extensor digitorum longus (EDL) and soleus (SOL) muscle were ascertained by quantitative Western blotting to better understand their respective capabilities and limitations. HSP72 content of EDL and SOL muscle was only ～1.1 and 4.6 μmol/kg wet wt, respectively, and HSP25 content approximately twofold greater (～3.4 and ～8.9 μmol/kg, respectively). αB-crystallin content of EDL muscle was ～4.9 μmol/kg but in SOL muscle was ～30-fold higher (～140 μmol/kg). To examine fiber heterogeneity, HSP content was also assessed in individual fiber segments; every EDL type II fiber had less of each HSP than any SOL type I fiber, whereas the two SOL type II fibers examined were indistinguishable from the EDL type II fibers. Sarcolemma removal (fiber skinning) demonstrated that 10-20% of HSP25 and αB-crystallin was sarcolemma-associated in SOL fibers. HSP diffusibility was assessed from the extent and rate of diffusion out of skinned fiber segments. In unstressed SOL fibers, 70-90% of each HSP was readily diffusible, whereas ～95% remained tightly bound in fibers from SOL muscles heated to 45°C. Membrane disruption with Triton X-100 allowed dispersion of HSP72 and sarco(endo)plasmic reticulum Ca(2+)-ATPase pumps but did not alter binding of HSP25 or αB-crystallin. The amount of HSP72 in unstressed EDL muscle is much less than the number of its putative binding sites, whereas SOL type I fibers contain large amounts of αB-crystallin, suggesting its importance in normal cellular function without upregulation.     

Fiber number and type composition in extensor digitorum longus, soleus, and diaphragm muscles with aging in Fisher 344 rats

Histochemical (M-ATPase) fiber typing was done on extensor digitorum longus, (EDL), soleus (SOL), and diaphragm (DIA) muscles of barrier-reared Fisher 344 rats obtained at four different ages (3, 9, 28, and 30 months) from the colonies of the National Institute of Aging. In the EDL there are no differences in the percent of type I fibers among the four age groups. The percent of type IIa and IIb fibers also showed no difference between the 3 and 30 month age groups. There was no apparent trend for an increase or decrease in the percent of type IIa or IIb fibers between the four age groups. In both the SOL and DIA muscles the percent of type I fibers was greater in the aged than in the young groups. The percent of type IIa fibers was lower in the 30 month group than in the younger groups for both muscles. The percent of type IIb (DIA) and IIc (SOL) fibers did not change between groups. Total fiber number per cross section of muscle showed no change in the EDL over this age range or in the SOL after 9 months of age. These findings bring into question published results that imply that decreasing fiber number and preferential loss of type II (a and b) fibers are typical aging phenomena.     

Heart failure increases atrogin-1 and MuRF1 gene expression in skeletal muscle with fiber type-specific atrophy

Heart failure (HF) is characterized by a reduced tolerance to exercise due to early fatigue and dyspnea; this may be due in part to skeletal muscle myopathy with a shift from slow to fast fibers and loss of muscle mass. Muscle wasting does not occur similarly in all types of muscle fiber, thus we tested the hypothesis that HF induces skeletal muscle atrophy in a fiber type-specific manner altering the expression of atrogin-1 and MuRF1 in a fast muscle of rats with monocrotaline-induced heart failure. We studied extensor digitorum longus (EDL) muscle from both HF and control Wistar rats. Atrogin-1 and MuRF1 mRNA content were determined using Real-Time RT-qPCR while muscle fiber cross-sectional area (CSA) from sections stained histochemically for myofibrillar ATPase were used as an index of type-specific fiber atrophy. The measurement of gene expression by RT-qPCR revealed that EDL muscle mRNA expression of MuRF1 and atrogin-1 was significantly increased in the HF group. Muscle fiber type IIB CSA decreased in the HF group compared to the CT group; there was no significant difference in muscle fiber types I and IIA/D CSA between the HF and CT groups. In conclusion, we showed that HF induces fiber type IIB specific atrophy, up-regulating atrogin-1 and MuRF1 mRNA expression in EDL muscle of monocrotaline treated rats.     

Denervation produces different single fiber phenotypes in fast- and slow-twitch hindlimb muscles of the rat

Using a single, mechanically skinned fiber approach, we tested the hypothesis that denervation (0 to 50 days) of skeletal muscles that do not overlap in fiber type composition [extensor digitorum longus (EDL) and soleus (SOL) muscles of Long-Evans hooded rats] leads to development of different fiber phenotypes. Denervation (50 day) was accompanied by 1) a marked increase in the proportion of hybrid IIB/D fibers (EDL) and I/IIA fibers (SOL) from 30% to >75% in both muscles, and a corresponding decrease in the proportion of pure fibers expressing only one myosin heavy chain (MHC) isoform; 2) complex muscle- and fiber-type specific changes in sarcoplasmic reticulum Ca²⁺-loading level at physiological pCa 7.1, with EDL fibers displaying more consistent changes than SOL fibers; 3) decrease by 50% in specific force of all fiber types; 4) decrease in sensitivity to Ca²⁺, particularly for SOL fibers (by 40%); 5) decrease in the maximum steepness of the force-pCa curves, particularly for the hybrid I/IIA SOL fibers (by 35%); and 6) increased occurrence of biphasic behavior with respect to Sr²⁺ activation in SOL fibers, indicating the presence of both slow and fast troponin C isoforms. No fiber types common to the two muscles were detected at any time points (day 7, 21, and 50) after denervation. The results provide strong evidence that not only neural factors, but also the intrinsic properties of a muscle fiber, influence the structural and functional properties of a particular muscle cell and explain important functional changes induced by denervation at both whole muscle and single cell levels. mechanically skinned fibers; myosin heavy chain isoforms; lineage; sarcoplasmic reticulum; Ca²⁺; Sr²⁺ sensitivity; Long-Evans hooded rat     

Continuous testosterone administration prevents skeletal muscle atrophy and enhances resistance to fatigue in orchidectomized male mice

Androgens promote anabolism in skeletal muscle; however, effects on subsequent muscle function are less well defined because of a lack of reliable experimental models. We established a rigorous model of androgen withdrawal and administration in male mice and assessed androgen regulation of muscle mass, structure, and function. Adult C57Bl/6J male mice were orchidectomized (Orx) or sham-operated (Sham) and received 10 wk of continuous testosterone (T) or control treatment (C) via intraperitoneal implants. Mass, fiber cross-sectional area (CSA), and in vitro contractile function were assessed for fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles. After 10 wk, Orx+C mice had reduced body weight gain (P < 0.05), seminal vesicle mass (P < 0.01), and levator ani muscle mass (P < 0.001) compared with Sham+C mice, and these effects were prevented with testosterone treatment. Orx+T mice had greater EDL (P < 0.01) and SOL (P < 0.01) muscle mass compared with Orx+C mice; however, median fiber CSA was not significantly altered in these muscles. EDL and SOL muscle force was greater in Sham+T compared with Orx+C mice (P < 0.05) in proportion to muscle mass. Unexpectedly, Orx+T mice had increased fatigue resistance of SOL muscle compared with Orx+C mice (P < 0.001). We used a rigorous model of androgen withdrawal and administration in male mice to demonstrate an essential role of androgens in the maintenance of muscle mass and force. In addition, we showed that testosterone treatment increases resistance to fatigue of slow- but not fast-twitch muscle.     

Skeletal muscle adaptation in rats flown on Cosmos 1667

Seven male Wistar rats were subjected to 7 days of weightlessness on the Soviet biosatellite Cosmos 1667. Muscle histomorphometry and biochemical analyses were performed on the soleus (SOL) and extensor digitorum longus (EDL) of flight rats (group F) and compared with data from three groups of terrestrial controls: one subjected to conditions similar to group F in space except for the state of weightlessness (group S) and the others living free in a vivarium (V1, V2). Relative to group V2 (its age and weight-matched control group), group F showed a greater decrease of muscle mass in SOL (23%) than in EDL (11%). In SOL a decrease in the percentage of type I fibers was counterbalanced by a simultaneous increase in type IIa fibers. The cross-sectional area of type I fiber was reduced by 24%. No statistically significant difference in capillarization and enzymatic activities was observed between the groups. In EDL a reduction in type I fiber distribution and 3-hydroxyacyl-CoA-dehydrogenase activity (27%) occurred after the flight. The small histochemical and biochemical changes reported suggest the interest in studying muscular adaptation during a flight of longer duration.     

MHC-based fiber type and E-C coupling characteristics in mechanically skinned muscle fibers of the rat

In this study, we investigated whether the previously established differences between fast- and slow-twitch single skeletal muscle fibers of the rat, in terms of myosin heavy chain (MHC) isoform composition and contractile function, are also detectable in excitation-contraction (E-C) coupling. We compared the contractile responsiveness of electrophoretically typed, mechanically skinned single fibers from the soleus (Sol), the extensor digitorum longus (EDL), and the white region of the sternomastoid (SM) muscle to t-system depolarization-induced activation. The quantitative parameters assessed were the amplitude of the maximum depolarization-induced force response (DIFR(max); normalized to the maximum Ca(2+)-activated force in that fiber) and the number of responses elicited until the force declined by 75% of DIFR(max) (R-D(75%)). The mean DIFR(max) values for type IIB EDL and type IIB SM fibers were not statistically different, and both were greater than the mean DIFR(max) for type I Sol fibers. The mean R-D(75%) for type IIB EDL fibers was greater than that for type I Sol fibers as well as type IIB SM fibers. These data suggest that E-C coupling characteristics of mechanically skinned rat single muscle fibers are related to MHC-based fiber type and the muscle of origin.     

Catalase-positive microperoxisomes in rat soleus and extensor digitorum longus muscle fiber types

The size, distribution, and content of catalase-reactive microperoxisomes were studied cytochemically in slow-twitch oxidative (SO), fast-twitch oxidative glycolytic (FOG), and fast-twitch glycolytic (FG) fibers of soleus and extensor digitorum longus (EDL) rat muscles. Fiber types were classified on the basis of mitochondrial content and distribution, Z-band widths, and myofibril size and shape. Microperoxisomes were generally located between myofibrils at the I-bands. The absence of crystalloid inclusions prevented positive identification of microperoxisomes in nonreacted and aminotriazole-inhibited muscles. EDL and soleus SO fibers possessed the largest microperoxisomes, whereas FOG and FG fibers of the EDL contained small- to medium-sized microperoxisomes. Comparing either microperoxisome number per muscle fiber area or microperoxisome area per fiber area revealed significant differences between fiber types with this ranking: soleus SO greater than EDL SO greater than EDL FOG greater than EDL FG. The present observations demonstrate that the content of catalase-positive microperoxisomes is greatest in the oxidative muscle fiber types. These cytochemical findings account for the higher catalase activity in homogenates of soleus muscles as compared to that of EDL muscles, because the soleus contains more oxidative fibers than EDL.     

Effects of β-hydroxy-β-methylbutyrate treatment in different types of skeletal muscle of intact and septic rats

β-Hydroxy-β-methylbutyrate (HMB) is a leucine metabolite that may have a positive effect in protein catabolic conditions. Therefore, we hypothesized that HMB treatment could attenuate the sepsis-induced protein catabolic state. The aims of our study were to elucidate the effect of HMB in healthy and septic animals and to evaluate the differences in the action of HMB in different muscle types. Intact and septic (5 mg endotoxin/kg i.p.) rats were administered with HMB (0.5 g/kg/day) or saline. After 24 h, extensor digitorum longus (EDL) and soleus (SOL) muscles were isolated and used for determination of total and myofibrillar proteolysis, protein synthesis, leucine oxidation, activity of cathepsins B and L, chymotrypsin-like activity, and expression of α-subunits of proteasome. Our results indicate that the catabolic state induced by the endotoxin treatment was caused both by increase in protein breakdown (due to activation of proteasome system) and by attenuation of protein synthesis. The EDL (muscle composed of white, fast-twitch fibers) was more susceptible to these changes than the SOL (muscle composed of red, slow-twitch fibers). The HMB treatment had no effect in healthy animals but counteracted the changes in septic animals. The action of HMB was mediated by attenuation of proteasome activity and protein breakdown, not by stimulation of protein synthesis. More pronounced effect of the HMB treatment on myofibrillar proteolysis was observed in the SOL.     

Interactions between intrinsic regulation and neural modulation of acetylcholinesterase in fast and slow skeletal muscles

1. Initiation of subsynaptic sarcolemmal specialization and expression of different molecular forms of AChE were studied in fast extensor digitorum longus (EDL) and slow soleus (SOL) muscle of the rat under different experimental conditions in order to understand better the interplay of neural influences with intrinsic regulatory mechanisms of muscle cells. 2. Former junctional sarcolemma still accumulated AChE and continued to differentiate morphologically for at least 3 weeks after early postnatal denervation of EDL and SOL muscles. In noninnervated regenerating muscles, postsynaptic-like sarcolemmal specializations with AChE appeared (a) in the former junctional region, possibly induced by a substance in the former junctional basal lamina, and (b) in circumscribed areas along the whole length of myotubes. Therefore, the muscle cells seem to be able to produce a postsynaptic organization guiding substance, located in the basal lamina. The nerve may enhance the production or accumulation of this substance at the site of the future motor end plate. 3. Significant differences in the patterns of AChE molecular forms in EDL and SOL muscles arise between day 4 and day 10 after birth. The developmental process of downregulation of the asymmetric AChE forms, eliminating them extrajunctionally in the EDL, is less efficient in the SOL. The presence of these AChE forms in the extrajunctional regions of the SOL correlates with the ability to accumulate AChE in myotendinous junctions. The typical distribution of the asymmetric AChE forms in the EDL and SOL is maintained for at least 3 weeks after muscle denervation. 4. Different patterns of AChE molecular forms were observed in noninnervated EDL and SOL muscles regenerating in situ. In innervated regenerates, patterns of AChE molecular forms typical for mature muscles were instituted during the first week after reinnervation. 5. These results are consistent with the hypothesis that intrinsic differences between slow and fast muscle fibers, concerning the response of their AChE regulating mechanism to neural influences, may contribute to different AChE expression in fast and slow muscles, in addition to the influence of different stimulation patterns.     

废用条件下大鼠和达乌尔黄鼠骨骼肌氧化应激和抗氧化防御能力与肌萎缩的比较研究

有鳞类(蛇和蜥蜴)具有较发达的嗅器和犁鼻器,对其不同种类嗅觉结构的认识有助于阐明爬行动物化学感觉的进化。本文采用组织学方法比较了草原沙蜥(Phrynocephalus frontalis)、荒漠沙蜥(P. przewalskii)、密点麻蜥(Eremias multiocellata)和秦岭滑蜥(Scincella tsinlingensis)的嗅器及犁鼻器。结果发现,草原沙蜥的鼻腔较为狭长,秦岭滑蜥呈梨形,其他两种蜥蜴的鼻腔略成圆形。秦岭滑蜥的嗅上皮最厚,其次是密点麻蜥和草原沙蜥,荒漠沙蜥最薄。犁鼻器主要由犁鼻腔、犁鼻感觉上皮、犁鼻神经及蘑菇体等组成,没有腺体。草原沙蜥和荒漠沙蜥的犁鼻腔较为宽阔,密点麻蜥和秦岭滑蜥的较窄。4种蜥蜴的犁鼻感觉上皮均较嗅上皮厚,蘑菇体向后逐渐缩小至消失,犁鼻感觉上皮成闭环状,包围犁鼻腔。密点麻蜥和秦岭滑蜥的犁鼻感觉上皮位于犁鼻器的背侧,蘑菇体位于腹侧;与此不同,两种沙蜥的犁鼻感觉上皮偏向于犁鼻器的腹内侧,蘑菇体位于背外侧。密点麻蜥的犁鼻感觉上皮最厚,其次为秦岭滑蜥,两种沙蜥最薄;秦岭滑蜥犁鼻感觉上皮的感觉细胞密度最高,其次是密点麻蜥,两种沙蜥最低。这些结果提示,密点麻蜥和秦岭滑蜥对嗅觉信号的依赖和投入较两种沙蜥多;4种蜥蜴犁鼻器的结构差异间接地佐证了有鳞类犁鼻器系统发生的特异性。     

超排处理对雌兔生殖器官中表皮生长因子表达的影响

本研究采用免疫荧光组织化学染色法和蛋白免疫印迹法比较研究了后肢去负荷大鼠(Rattus norvegicus)和冬眠不活动达乌尔黄鼠(Spermophilus dauricus)不同类型骨骼肌氧化应激水平和抗氧化防御能力及与肌萎缩之间的关系。结果显示,后肢去负荷14 d后,大鼠比目鱼肌和趾长伸肌肌萎缩程度显著升高,过氧化氢和丙二醛水平增加,Nrf2介导的抗氧化信号通路及下游抗氧化酶蛋白表达及活性显著下降;而冬眠不活动达乌尔黄鼠骨骼肌中肌萎缩指标并未出现变化,氧化应激水平维持夏季组水平,抗氧化酶和调控因子出现不同程度升高。研究表明,后肢去负荷导致非冬眠大鼠骨骼肌氧化应激水平升高,抗氧化防御能力减弱,可能是导致大鼠废用性肌萎缩的重要机制之一;而冬眠动物达乌尔黄鼠骨骼肌在自然废用状态下,抗氧化防御能力增强可能是防止自然冬眠不活动引起的废用性肌萎缩的重要机制。     

Fiber-type-specific alphaB-crystallin distribution and its shifts with T(3) and PTU treatments in rat hindlimb muscles.

Changes in alphaB-crystallin content in adult rat soleus and extensor digitorum longus (EDL) were examined after 8 wk of 3,5, 3'-triiodothyronine (T(3)) and propylthiouracil (PTU) treatments. Cellular distributions of alphaB-crystallin expression related to fiber type, and distribution shifts with these treatments were also examined in detail from the gray level of reactivity to specific anti-alphaB-crystallin antibody. alphaB-crystallin content in both soleus and EDL muscles was significantly decreased after T(3), and that in EDL was significantly increased over twofold after PTU treatment. In both control soleus and EDL muscles, the gray level of type I fibers was higher than that of type II fibers. alphaB-crystallin expression among type II subtypes was muscle specific; the order was type I > IIa > IIx > IIb in control EDL muscle and type IIx > or = IIa in soleus muscle. The relation was basically unchanged in both muscles after T(3) treatment and was, in particular, well maintained in EDL muscle. Under hypothyroidism conditions with PTU, the mean alphaB-crystallin levels of type IIa and IIx fibers were significantly lower than levels under control conditions. Thus the relation between fiber type and the expression manner of stress protein alphaB-crystallin is muscle specific and also is well regulated under thyroid hormone, especially in fast EDL muscle.     

The effects of 10 days of spaceflight on the shuttle Endeavour on predominantly fast-twitch muscles in the rat

The primary purpose of this investigation was to determine the effects of microgravity on muscle fibers of the predominantly fast-twitch muscles in the rat. Cross sectional area and myosin heavy chain (MHC) composition were assessed in order to establish the acute effects of microgravity associated with spaceflight. The extensor digitorum longus (EDL) and gastrocnemius muscles were removed from 12 male Fisher 344 rats which had undergone 10 days of spaceflight aboard the space shuttle Endeavor and from 12 age- and weight-matched control animals. Both groups of animals received similar amounts of food and water and were synchronized for photoperiods, environmental temperature, and humidity. Significant (P < 0.05) reductions in muscle fiber size were observed in the gastrocnemius (fiber types I, IIA, IIDB, and IIB) and EDL (fiber type IIB) muscles after spaceflight. Significant MHC isoform transformations also resulted during this brief period of microgravity exposure with a significant decrease in MHC IId isoform in the EDL muscle. A significant decrease was also observed in the MHC IId isoform in the superficial (white) component of the gastrocnemius muscle after spaceflight, although no alterations in MHC profile were demonstrated in the deep (red) component of this muscle. These findings highlight the rapid plasticity of skeletal muscle during short-term spaceflight. If such pronounced adaptations to spaceflight also occur in humans, then astronauts are likely to suffer severe decrements in skeletal muscle performance with long-term space flight and upon return to earth after both short- and long-term missions. Thus, countermeasures aimed at slowing or even preventing muscle fiber atrophy are warranted.     

Differential morphofunctional characteristics and gene expression in fast and slow muscle of rats with monocrotaline-induced heart failure

Heart failure (HF) is characterized by limited exercise tolerance, skeletal muscle atrophy, a shift toward fast muscle fiber, and myogenic regulatory factor (MRF) changes. Reactive oxygen species (ROS) also contribute to target organ damage in this syndrome. In this study, we investigated and compared morphofunctional characteristics and gene expression in Soleus (SOL--oxidative and slow twitching muscle) and in Extensor Digitorum Longus (EDL--glycolytic and fast twitching muscle) during HF. Two groups of rats were used: control (CT) and heart failure (HF), induced by a single injection of monocrotaline. MyoD and myogenin gene expression were determined by RT-qPCR, and MHC isoforms by SDS-PAGE; muscle fiber type frequency and cross sectional area (CSA) were analyzed by mATPase. A biochemical study was performed to determine lipid hydroperoxide (LH), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD); myography was used to determine amplitude, rise time, fall time, and fatigue resistance in both muscles. HF showed SOL and EDL muscle atrophy in all muscle fiber types; fiber frequency decreased in type IIC and muscle contraction fall time increased only in SOL muscle. Myogenin mRNA expression was lower in SOL and myoD decreased in HF EDL muscle. LH increased, and SOD and GSH-Px activity decreased only in HF SOL muscle. HF EDL muscle did not present changes in MHC distribution, contractile properties, HL concentration, and antioxidant enzyme activity. In conclusion, our results indicate that monocrotaline induced HF promoted more prominent biochemical, morphological and functional changes in SOL (oxidative and slow twitching muscle). Although further experiments are required to better determine the mechanisms involved in HF pathophysiology, our results contribute to understanding the muscle-specific changes that occur in this syndrome.     

Influences of temperature, oxidative stress, and phosphorylation on binding of heat shock proteins in skeletal muscle fibers

Heat shock proteins (HSPs) help maintain cellular function in stressful situations, but the processes controlling their interactions with target proteins are not well defined. This study examined the binding of HSP72, HSP25, and αB-crystallin in skeletal muscle fibers following various stresses. Rat soleus (SOL) and extensor digitorum longus (EDL) muscles were subjected in vitro to heat stress or strongly fatiguing stimulation. Superficial fibers were "skinned" by microdissection and HSP diffusibility assessed from the extent of washout following 10- to 30 min exposure to a physiological intracellular solution. In fibers from nonstressed (control) SOL muscle, >80% of each HSP is readily diffusible. However, after heating a muscle to 40°C for 30 min ～95% of HSP25 and αB-crystallin becomes tightly bound at nonmembranous myofibrillar sites, whereas HSP72 bound at membranous sites only after heat treatment to ≥44°C. The ratio of reduced to oxidized cytoplasmic glutathione (GSH:GSSG) decreased approximately two- and fourfold after heating muscles to 40° and 45°C, respectively. The reducing agent dithiothreitol reversed HSP72 binding in heated muscles but had no effect on the other HSPs. Intense in vitro stimulation of SOL muscles, sufficient to elicit substantial oxidation-related loss of maximum force and approximately fourfold decrease in the GSH:GSSG ratio, had no effect on diffusibility of any of the HSPs. When skinned fibers from heat-treated muscles were bathed with additional exogenous HSP72, total binding increased approximately two- and 10-fold, respectively, in SOL and EDL fibers, possibly reflective of the relative sarco(endo)plasmic reticulum Ca(2+)-ATPase pump densities in the two fiber types. Phosphorylation at Ser59 on αB-crystallin and Ser85 on HSP25 increased with heat treatment but did not appear to determine HSP binding. The findings highlight major differences in the processes controlling binding of HSP72 and the two small HSPs. Binding was not directly related to cytoplasmic oxidative status, but oxidation of cysteine residues influenced HSP72 binding.     

The effect of a unilateral muscle transplantation on the muscle fiber type and the MyHC isoform content in unoperated hind limb slow and fast muscles of the inbred Lewis rats

To reveal the effect of foreign innervation and altered thyroid status on fiber type composition and the myosin heavy chain (MyHC) isoform expression in the rat slow soleus (SOL) and fast extensor digitorum longus (EDL) muscles, a method of heterochronous isotransplantation was developed. In this experimental procedure, the SOL or EDL muscles of young inbred Lewis rats are grafted either into the host EDL or SOL muscles of adult rats of the same strain with normal or experimentally altered thyroid status. To estimate the extent of fiber type transitions in the transplanted muscles, the SOL and EDL muscle from the unoperated leg and unoperated muscles from the operated leg could be legitimately used as controls, but only when the experimental procedure itself does not affect these muscles. To verify this assumption, we have compared the fiber type composition and the MyHC isoform content of unoperated contralateral SOL and EDL muscles and ipsilateral unoperated SOL muscle of experimental rats after unilateral isotransplantation into the host EDL muscle with corresponding muscles of the naive rats of the same age and strain. We provide compelling evidence that the unilateral heterochronous isotransplantation has no significant effect on the fiber type composition and the MyHC isoform content of unoperated muscles of experimental animals. Hence, these muscles can be used as controls in our grafting experiments.