Silencing SERCA1b in a few fibers stimulates growth in the entire regenerating soleus muscle

The neonatal isoform of the sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase 1 (SERCA1b) is a dominant Ca²⁺ pump in the young fibers of regenerating muscle. In vivo transfection of about 1% of the fibers with SERCA1b RNAi plasmid resulted in no apparent change in the transfected fibers, but enhanced the increase of fresh weight and fiber size in the whole regenerating rat soleus muscle, until the normal size was reached. Co-transfection of calcineurin inhibitor cain/cabin-1 with SERCA1b RNAi was sufficient to cut down the widespread growth stimulation, but the subsequent transfection of cain into the SERCA1b RNAi transfected muscle did not inhibit muscle growth. The SERCA1b RNAi preferably upregulated the expression of the NFAT reporter lacZ compared to controls when co-transfected into the fibers. Notably, perimuscular injection of interleukin-4 (IL-4) antibody but not that of an unrelevant antibody completely abolished the growth-promoting effect of SERCA1b RNAi. This indicates that silencing SERCA1b in a few fibers stimulates the calcineurin-NFAT-IL-4 pathway and fiber growth in the whole regenerating soleus. These results suggest the presence of an autocrine–paracrine coordination of growing muscle fibers, and put forward a new method to stimulate skeletal muscle regeneration.     

Expression of myosin isoforms during notexin-induced regeneration of rat soleus muscles

Myosin isozymes and their fiber distribution were studied during regeneration of the soleus muscle of young adult (4-6 week old) rats. Muscle degeneration and regeneration were induced by a single subcutaneous injection of a snake toxin, notexin. If reinnervation of the regenerating muscle was allowed to occur (functional innervation nearly complete by 7 days), then fiber diameters continued to increase and by 28 days after toxin treatment they attained the same values as fibers in the contralateral soleus. If the muscles were denervated at the time of toxin injection, the early phases of regeneration still took place but the fibers failed to continue to increase in size. Electrophoresis of native myosin showed multiple bands between 3 and 21 days of regeneration which could be interpreted as indicating the presence of embryonic, neonatal, fast and slow myosins in the innervated muscles. Adult slow myosin became the exclusive from in innervated regenerates. In contrast, adult fast myosin became the predominant form in denervated regenerating muscles. Immunocytochemical localization of myosin isozymes demonstrated that in innervated muscles the slow form began to appear in a heterogeneous fashion at about 7 days, and became the major form in all fibers by 21-28 days. Thus, the regenerated muscle was almost entirely composed of slow fibers, in clear contrast to the contralateral muscle which was still substantially mixed. In denervated regenerating muscles, slow myosin was not detected biochemically or immunocytochemically whereas fast myosin was detected in all denervated fibers by 21-28 days. The regenerating soleus muscle therefore is clearly different from the developing soleus muscle in that the former is composed of a uniform fiber population with respect to myosin transitions. Moreover the satellite cells which account for the regeneration process in the soleus muscle do not appear to be predetermined with respect to myosin heavy chain expression, since the fibers they form can express either slow or fast isoforms. The induction of the slow myosin phenotype is entirely dependent on a positive, extrinsic influence of the nerve.     

Isoform switching in myofibrillar and excitation-contraction coupling proteins contributes to diminished contractile function in regenerating rat soleus muscle.

Postnatal development of skeletal muscle occurs through the progressive transformation of diverse biochemical, metabolic, morphological, and functional characteristics from the embryonic to the adult phenotype. Since muscle regeneration recapitulates postnatal development of muscle fiber, it offers an appropriate experimental model to investigate the existing relationships between diverse muscle functions and the expression of key protein isoforms, particularly at the single-fiber level. This study was carried out in regenerating soleus muscle 14 days after injury. At this intermediate stage, the regenerating muscle exhibited a recovery of mass greater than its force generation capacity. The lower specific tension of regenerating muscle suggested intrinsic defective excitation-contraction coupling and/or contractility processes. The presence of developmental isoforms of both the voltage-gated Ca(2+) channel (alpha(1)C) and of ryanodine receptor 3, paralleled by an abnormal caffeine contracture development, confirms the immature excitation-contraction coupling of the regenerating muscle. The defective Ca(2+) handling could also be confirmed by the lower sarcoplasmic reticulum caffeine sensitivity of regenerating single fibers. Also, regenerating single fibers revealed a lower maximal specific tension, which was associated with the residual presence of embryonic myosin heavy chains. Moreover, the fibers showed a reduced Ca(2+) sensitivity of myofibrillar proteins, particularly those simultaneously expressing the slow and fast isoforms of troponin C. The present results indicate that the expression of developmental proteins determines the incomplete functional recovery of regenerating soleus.     

Effects of mechanical over-loading on the properties of soleus muscle fibers, with or without damage, in wild type and mdx mice

Effects of mechanical over-loading on the characteristics of regenerating or normal soleus muscle fibers were studied in dystrophin-deficient (mdx) and wild type (WT) mice. Damage was also induced in WT mice by injection of cardiotoxin (CTX) into soleus muscle. Over-loading was applied for 14 days to the left soleus muscle in mdx and intact and CTX-injected WT mouse muscles by ablation of the distal tendons of plantaris and gastrocnemius muscles. All of the myonuclei in normal muscle of WT mice were distributed at the peripheral region. But, central myonuclei were noted in all fibers of WT mice regenerating from CTX-injection-related injury. Further, many fibers of mdx mice possessed central myonuclei and the distribution of such fibers was increased in response to over-loading, suggesting a shift of myonuclei from peripheral to central region. Approximately 1.4% branched fibers were seen in the intact muscle of mdx mice, although these fibers were not detected in WT mice. The percentage of these fibers in mdx, not in WT, mice was increased by over-loading (～51.2%). The fiber CSA in normal WT mice was increased by over-loading (p<0.05), but not in mdx and CTX-injected WT mice. It was suggested that compensatory hypertrophy is induced in normal muscle fibers of WT mice following functional over-loading. But, it was also indicated that muscle fibers in mdx mice are susceptible to mechanical over-loading and fiber splitting and shift of myonuclei from peripheral to central region are induced.     

dnRas stimulates autocrine-paracrine growth of regenerating muscle via calcineurin-NFAT-IL-4 pathway

Ras and calcineurin are members of two independent pathways in muscle growth but their interaction is not known. This work shows that the transfection of about 1% of the muscle fibers with dominant negative Ras (dnRas) shows a wilder effect; it stimulates the fiber growth in the entire regenerating soleus muscle, including the nontransfected fibers. Co-transfection with the calcineurin inhibitor cain/cabin prevented the growth stimulation. Injection of antibody for interleukin-4 (IL-4) also abolished the growth ameliorating effect. These results suggest that the inactivation of Ras in 1% of the fibers upregulates the calcineurin-NFAT-IL-4 pathway and the secreted IL-4 triggers fiber growth stimulation in the whole regenerating soleus muscle of the rat. The results highlight the importance of the autocrine-paracrine regulation in muscle regeneration and hint to a novel method of gene theraphy of degenerative-regenerative muscle dystrophies.     

Optimizing hyaluronidase dose and plasmid DNA delivery greatly improves gene electrotransfer efficiency in rat skeletal muscle

Transfection of rat skeletal muscle in vivo is a widely used research model. However, gene electrotransfer protocols have been developed for mice and yield variable results in rats. We investigated whether changes in hyaluronidase pre-treatment and plasmid DNA delivery can improve transfection efficiency in rat skeletal muscle. We found that pre-treating the muscle with a hyaluronidase dose suitable for rats (0.56 U/g b.w.) prior to plasmid DNA injection increased transfection efficiency by >200% whereas timing of the pre-treatment did not affect efficiency. Uniformly distributing plasmid DNA delivery across the muscle by increasing the number of plasmid DNA injections further enhanced transfection efficiency whereas increasing plasmid dose from 0.2 to 1.6 µg/g b.w. or vehicle volume had no effect. The optimized protocol resulted in ~80% (CI95%: 79–84%) transfected muscle fibers with a homogenous distribution. We also show that transfection was stable over five weeks of regular exercise or inactivity. Our findings show that species-specific plasmid DNA delivery and hyaluronidase pre-treatment greatly improves transfection efficiency in rat skeletal muscle.     

Respective effects of anabolic/androgenic steroids and physical exercise on isometric contractile properties of regenerating skeletal muscles in the rat

We examined the respective effects of anabolic-androgenic steroids and physical exercise on the contractile properties of regenerating fast and slow hindlimb skeletal muscles. Degeneration/regeneration of the left extensor digitorum longus muscles (EDL) and soleus of young Wistar male rats was induced by a snake venom (Notechis scutatus scutatus) injection. During muscle regeneration, experimental rats were either treated with nandrolone (NAN, nortestosterone, im, 2 mg X kg(-1) X week(-1), or endurance exercised on a treadmill (EXE, 60 min x day(-1), 10-40 m X min(-1). Twenty-one days after injury, isometric contractile properties of regenerating muscles were studied in situ. Neither the nandrolone treatment nor the physical exercise program was able to change significantly muscle contraction parameters both in twitch and tetanus in both regenerating EDL and soleus (p > 0.05). However, we observed a greater peak twitch tension in NAN versus grouped control and EXE EDL (p < 0.01). In conclusion, endurance exercise program or anabolic-androgenic steroid (nortestosterone) treatment did not significantly improve isometric contractile properties of regenerating slow and fast muscles in the male young rats.     

Expression of nestin,desmin and vimentin in intact and regenerating muscle spindles of rat hind limb skeletal muscles

We describe the expression and distribution patterns of nestin, desmin and vimentin in intact and regenerating muscle spindles of the rat hind limb skeletal muscles. Regeneration was induced by intramuscular isotransplantation of extensor digitorum longus (EDL) or soleus muscles from 15-day-old rats into the EDL muscle of adult female inbred Lewis rats. The host muscles with grafts were excised after 7-, 16-, 21- and 29-day survival and immunohistochemically stained. Nestin expression in intact spindles in host muscles was restricted to Schwann cells of sensory and motor nerves. In transplanted muscles, however, nestin expression was also found in regenerating “spindle fibers”, 7 and 16 days after grafting. From the 21st day onwards, the regenerated spindle fibers were devoid of nestin immunoreactivity. Desmin was detected in spindle fibers at all developmental stages in regenerating as well as in intact spindles. Vimentin was expressed in cells of the outer and inner capsules of all muscle spindles and in newly formed myoblasts and myotubes of regenerating spindles 7 days after grafting. Our results show that the expression pattern of these intermediate filaments in regenerating spindle fibers corresponds to that found in regenerating extrafusal fibers, which supports our earlier suggestion that they resemble small-diameter extrafusal fibers.     

Expression of SERCA2a is not regulated by calcineurin or upon mechanical unloading in skeletal muscle regeneration

This study investigates to what extent the expression of the slow myosin heavy chain (MyHCI) isoform and the slow type sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) isoform are co-regulated in fibers of regenerating skeletal soleus muscle. Both overexpression of cain, a calcineurin inhibitor, or partial tenotomy prevented the expression of MyHCI but left SERCA2a expression unaffected in fibers of regenerating soleus muscles. These data complement those from different experimental models and clearly show that the expression of MyHCI and SERCA2a--the major proteins mediating, respectively, the slow type of contraction and relaxation--are not coregulated in regenerating soleus muscle.     

Length-tension relationships are altered in regenerating muscles of the rat after bupivacaine injection.

Intramuscular injection of bupivacaine causes complete degeneration of fibers in extensor digitorum longus (EDL) muscles of rats, followed by complete regeneration within 60 days. Previous studies have shown that regenerated EDL muscles are protected from contraction-induced injury 60 days after bupivacaine injection. It is possible that these regenerated muscles have altered length-tension relations because of fiber remodeling. We tested the hypothesis that length-tension relations are different in bupivacaine-injected and noninjected control muscles. EDL and soleus muscles of the right hindlimb of deeply anesthetized rats were injected with bupivacaine and then allowed to recover for 7, 14, 21, or 60 days (7D, 14D, 21D, 60D), and isometric contractile properties were assessed. Muscles of the contralateral limb were not injected and served as control. EDL muscles recovered from bupivacaine injection more rapidly than soleus muscles, with mass restored to control levels at 21D, and isometric tetanic force (P(o)) restored to control at 60D. In contrast, mass and P(o) of injected soleus muscles was not restored to control even at 60D. In 7D EDL muscles, length-tension curves were shifted leftward compared with control, but in 21D and 60D EDL muscles length-tension curves were right shifted significantly (treatment x muscle length: P < 0.001). Although no clear shift in the position of the length-tension curve was observed in regenerating soleus muscles, force production was enhanced on the descending limb of the curve in 60D soleus muscles (treatment x relative muscle length: P < 0.01). The rightward shift in the length-tension curve of EDL muscles 60 days after bupivacaine injection is likely to contribute to the mechanism for their previously observed protection from contraction-induced injury.     

BASAL LAMINA: THE SCAFFOLD FOR ORDERLY CELL REPLACEMENT : Observations on Regeneration of Injured Skeletal Muscle Fibers and Capillaries

To explore in detail the relationships between basal lamina (BL) and regenerating cells, we have studied the reconstruction of skeletal muscle fibers and their associated capillaries in portions of rat and rabbit skeletal muscles after injury with either freezing, ischemia, or in situ autografting. Each type of injury produces complete necrosis of cells. The BL, however, remains intact in the area of injury and maintains a "map" of the outline of the spatial relationships between muscle fibers and capillaries. Repopulation of the defect with new cells occurs primarily along the old BL. The spatial relationship between cells, as it existed before injury, is thus reestablished. This process appears to be aided by the ability of each category of regenerating cells to grow along the cell-supporting surface of its own BL. The regenerating cells of muscle fibers and capillaries frequently form a new layer of BL. It is of the usual thickness and is deposited primarily along the outer surfaces of plasma membranes in locations in which the new cells are separated from the old BL. Where an old layer of BL is present overlying a newly formed layer, the old layer may be retained or it may be removed. Removal of redundant BL is probably mediated by interstitial cells which embrace the outside surfaces of BL of regenerated skeletal muscle fibers and capillaries.     

Effects of exogenous growth hormone on skeletal muscle of young female rats

We examined the effects of exogenous growth hormone (GH) treatment on the soleus and rectus femoris muscles of young female rats. Rat GH (1.8 IU/mg) was administered for 3 weeks by subcutaneous injection, twice a day, at doses of 0.5, 0.6, and 0.8 mg/day during the 1st, 2nd, and 3rd week, respectively. Final body weight, as well as wet and dry weight, of the soleus and rectus femoris muscles were significantly greater in the GH-treated group, compared to controls. Muscle weight to body weight ratios did not differ between the two groups. The fiber type composition of the soleus muscle was determined by histochemical staining for myosin ATPase activity. No statistically significant difference was found between the GH-treated and the control groups in the percentages of fiber types. However, GH treatment significantly increased the cross-sectional area of type II fibers of the soleus muscle. These results suggest that, in young female rats, acceleration of body weight gain by homologous GH administration is accompanied by a proportional hypertrophy of skeletal muscle mass. Increased muscle mass is due to hypertrophy of muscle fibers. Type II muscle fibers appear to be more sensitive to GH stimulation.     

Enthalpy efficiency of the soleus muscle contributes to improvements in running economy

During human running, the soleus, as the main plantar flexor muscle, generates the majority of the mechanical work through active shortening. The fraction of chemical energy that is converted into muscular work (enthalpy efficiency) depends on the muscle shortening velocity. Here, we investigated the soleus muscle fascicle behaviour during running with respect to the enthalpy efficiency as a mechanism that could contribute to improvements in running economy after exercise-induced increases of plantar flexor strength and Achilles tendon (AT) stiffness. Using a controlled longitudinal study design (n = 23) featuring a specific 14-week muscle–tendon training, increases in muscle strength (10%) and tendon stiffness (31%) and reduced metabolic cost of running (4%) were found only in the intervention group (n = 13, p < 0.05). Following training, the soleus fascicles operated at higher enthalpy efficiency during the phase of muscle–tendon unit (MTU) lengthening (15%) and in average over stance (7%, p < 0.05). Thus, improvements in energetic cost following increases in plantar flexor strength and AT stiffness seem attributed to increased enthalpy efficiency of the operating soleus muscle. The results further imply that the soleus energy production in the first part of stance, when the MTU is lengthening, may be crucial for the overall metabolic energy cost of running.     

Targeting of calsequestrin to the sarcoplasmic reticulum of skeletal muscle upon deletion of its glycosylation site

The glycoprotein calsequestrin (CS) is segregated to the junctional sarcoplasmic reticulum (jSR) and is responsible for intraluminal Ca(2+) binding. A chimeric CS-hemoagglutinin 1 (HA1), obtained by adding the nine amino acid viral epitope hemoagglutinin to the carboxy terminal of CS and shown to be correctly segregated to skeletal muscle jSR [A. Nori, K. A. Nadalini, A. Martini, R. Rizzuto, A. Villa, and P. Volpe (1997). Chimeric calsequestrin and its targeting to the junctional sarcoplasmic reticulum of skeletal muscle. Am. J. Physiol. 272, C1420-C1428] lends itself as a molecular tool to investigate the targeting domains of CS. A putative targeting mechanism of CS to jSR implies glycosylation-dependent steps in the endoplasmic reticulum (ER) and Golgi complex. To test this hypothesis, CS-HA1DeltaGly, a mutant in which the unique N-glycosylation site Asn316 was changed to Ile, was engineered by site-directed mutagenesis. The mutant cDNA was transiently transfected in either HeLa cells, myoblasts of rat skeletal muscle primary cultures, or regenerating soleus muscle fibers of adult rats. The expression and intracellular localization of CS-HA1DeltaGly was studied by double-labeling epifluorescence by means of antibodies against either CS, HA1, or the ryanodine receptor calcium release channel. CS-HA1DeltaGly was expressed and retained to ER and ER/sarcoplasmic reticulum of HeLa cells and myotubes, respectively, and expressed, sorted, and correctly segregated to jSR of regenerating soleus muscle fibers. Thus, the targeting mechanism of CS in vivo appears not to be affected by glycosylation-that is, the sorting, docking, and segregation of CS are independent of cotranslational and posttranslational glycosylation or glycosylations.     

Denervation and reinnervation of fast and slow muscles. A histochemical study in rats.

A histochemical study, using myosin-adenosine triphosphatase activity at pH 9.4, was conducted in soleus and plantaris muscles of adult rats, after bilateral crushing of the sciatic nerve at the sciatic notch. The changes in fiber diameter and per cent composition of type I and type II fibers plus muscle weights were evaluated along the course of denervation-reinnervation curve at 1, 2, 3, 4 and 6 weeks postnerve crush. The study revealed that in the early denervation phase (up to 2 weeks postcrush) both the slow and fast muscles, soleus and plantaris, resepctively, atrophied similarly in muscle mass. Soleus increased in the number of type II fibers, which may be attributed to "disuse" effect. During the same period, the type I fibers of soleus atrophied as much or slightly more than the type II fibers; whereas the type II fibers of plantaris atrophied significantly more than the type I fibers, reflecting that the process of denervation, in its early stages, may affect the two fiber types differentially in the slow and fast muscles. It was deduced that the type I fibers of plantaris may be essentially different in the slow (soleus) and fast (plantaris) muscles under study. The onset of reinnervation, as determined by the increase in muscle weight and fiber diameter of the major fiber type, occurred in soleus and plantaris at 2 and 3 weeks postcrush, respectively, which confirms the earlier hypotheses that the slow muscles are reinnervated sooner than the fast muscles. It is suggested that the reinnervation of muscle after crush injury may be specific to the muscle type or its predominant fiber type.     

Effects of heat stress on muscle mass and the expression levels of heat shock proteins and lysosomal cathepsin L in soleus muscle of young and aged mice

Effects of heat stress on skeletal muscle mass in young and aged mice were investigated. Young (7-week) and aged (106-week) male C57BL/6J mice were randomly assigned to control and heat-stressed groups in each age. Mice in heat-stressed group were exposed to heat stress (41?°C for 60?min) in an incubator without anesthesia. Seven days after the exposure, soleus muscles were dissected from both hindlimbs. Protein content and the relative composition of Type II fibers in aged soleus were lower than those in young muscle. In aged soleus, higher baseline expression levels of HSP25, HSP72, and cathepsin L were observed compared with those in young muscle (p?相似文献   

Differential adaptation of growth and differentiation factor 8/myostatin, fibroblast growth factor 6 and leukemia inhibitory factor in overloaded, regenerating and denervated rat muscles

Mice genetically deficient in growth and differentiation factor 8 (GDF8/myostatin) had markedly increased muscle fiber numbers and fiber hypertrophy. In the regenerating muscle of mice possessing FGF6 mutation, fiber remodeling was delayed. Although myostatin and FGF6 may be important for the maintenance, regeneration and/or hypertrophy of muscle, little work has been done on the possible role of these proteins in adult muscle in vivo. Using Western blot and immunohistochemical analysis, we investigated, in rats, the distribution of myostatin, FGF6 and LIF proteins between slow- and fast-type muscles, and the adaptive response of these proteins in mechanically overloaded muscles, in regenerating muscles following bupivacaine injection and in denervated muscles after section of the sciatic nerve. The amounts of myostatin and LIF protein were markedly greater in normal slow-type muscles. In the soleus muscle, myostatin and LIF proteins were detected at the site of the myonucleus in both slow-twitch and fast-twitch fibers. In contrast, FGF6 protein was selectively expressed in normal fast-type muscles. Mechanical overloading rapidly enhanced the myostatin and LIF but not FGF6 protein level. In the regenerating muscles, marked diminution of myostatin and FGF6 was observed besides enhancement of LIF. Denervation of fast-type muscles rapidly increased the LIF, but decreased the FGF6 expression. Therefore, the increased expressions of myostatin and LIF play an important role in muscle hypertrophy following mechanical overloading. The marked reduction of FGF6 in the hypertrophied and regenerating muscle would imply that FGF6 regulates muscle differentiation but not proliferation of satellite cells and/or myoblasts.     

beta-Adrenoceptor signaling in regenerating skeletal muscle after beta-agonist administration

Stimulating the beta-adrenoceptor (beta-AR) signaling pathway can enhance the functional repair of skeletal muscle after injury, but long-term use of beta-AR agonists causes beta-AR downregulation, which may limit their therapeutic effectiveness. The aim was to examine beta-AR signaling during early regeneration in rat fast-twitch [extensor digitorum longus (EDL)] and slow-twitch (soleus) muscles after bupivacaine injury and test the hypothesis that, during regeneration, beta-agonist administration does not cause beta-AR desensitization. Rats received either the beta-AR agonist fenoterol (1.4 mgxkg(-1)xday(-1) ip) or saline for 7 days postinjury. Fenoterol reduced beta-AR density in regenerating soleus muscles by 42%. Regenerating EDL muscles showed a threefold increase in beta-AR density, and, again, these values were 43% lower with fenoterol treatment. An amplified adenylate cyclase (AC) response to isoproterenol was observed in cell membrane fragments from EDL and soleus muscles 7 days postinjury. Fenoterol attenuated this increase in regenerating EDL muscles but not soleus muscles. beta-AR signaling mechanisms were assessed using AC stimulants (NaF, forskolin, and Mn(2+)). Although beta-agonist treatment reduces beta-AR density in regenerating muscles, these muscles can produce large cAMP responses relative to healthy (uninjured) muscles. Desensitization of beta-AR signaling in regenerating muscles is prevented by altered rates of beta-AR synthesis and/or degradation, changes in G protein populations and coupling efficiency, and altered AC activity. These mechanisms have important therapeutic implications for modulating beta-AR signaling to enhance muscle repair after injury.     

Contractile properties,structure and fiber phenotype of intact and regenerating slow-twitch muscles of mice treated with cyclosporin A

We have studied the contractile properties, structure, fiber-type composition, and myosin heavy chain (MyHC) expression pattern of regenerating and intact soleus muscles of adult CBA/J mice treated with cyclosporin A (CsA) or vehicle solutions (Cremophor, saline). A comparison of muscles after 4-7 weeks drug application with those receiving vehicle showed that the isometric contractile force of intact drug-treated muscles was reduced (tetanus, -21%; twitch, -34%) despite normal mass and muscle cross-sectional area. The frequency of fast-twitch fibers was increased, whereas no innervation deficits, histopathological alterations, or changes in fiber numbers were observed. Regeneration after cryolesion of the contralateral soleus proceeded more slowly in CsA-treated than in vehicle-treated animals. Despite this, when muscle properties reached mature levels (4-7 weeks), muscle mass recovery was better in CsA-treated animals (30% higher weight, 50% more fiber profiles in cross-sections). The force production per unit cross-sectional area was deficient, but not the maximum tension. Twitch time-to-peak and half-relaxation time were shorter than controls correlating with a predominance of fast-twitch fibers (98% Type II fibers versus 16%-18% in control muscles) and fast MyHC isoforms. Partial reversal of this fast phenotype and an increase in muscle force were observed when the animals were left to recover without treatment for 5-8 weeks after CsA application over 7 weeks. The high numbers of fiber profiles in CsA-treated regenerated muscles and increased mass remained unchanged after withdrawal. Thus, CsA treatment has a hyperplastic effect on regenerating muscles, and drug-induced phenotype alterations are much more prominent in regenerated muscles.