Nerve activity controls fiber size and fiber type in skeletal muscle, but the underlying molecular mechanisms remain largely unknown. We have previously shown that Ras-mitogen-activated protein kinase and calcineurin control fiber type but not fiber size in regenerating rat skeletal muscle. Here we report that constitutively active protein kinase B (PKB), also known as Akt, increases fiber size and prevents denervation atrophy in regenerating and adult rat muscles but does not affect fiber type profile. The coexistence of hypertrophic muscle fibers overexpressing activated PKB with normal-size untransfected fibers within the same muscle points to a cell-autonomous control of muscle growth by PKB. The physiological role of this pathway is confirmed by the finding that PKB kinase activity and phosphorylation status are significantly increased in innervated compared with denervated regenerating muscles in parallel with muscle growth. Muscle fiber hypertrophy induced by activated PKB and by a Ras double mutant (RasV12C40) that activates selectively the phosphoinositide 3-kinase-PKB pathway is completely blocked by rapamycin, showing that the mammalian target of rapamycin kinase is the major downstream effector of this pathway in the control of muscle fiber size. On the other hand, nerve activity-dependent growth of regenerating muscle is only partially inhibited by dominant negative PKB and rapamycin, suggesting that other nerve-dependent signaling pathways are involved in muscle growth. The present results support the notion that fiber size and fiber type are regulated by nerve activity through different mechanisms.

Calcineurin (Cn) signaling has been implicated in nerve activitydependent fiber type specification in skeletal muscle, but the downstream effector pathway has not been established. We have investigated the role of the transcription factor nuclear factor of activated T cells (NFAT), a major target of Cn, by using an in vivo transfection approach in regenerating and adult rat muscles. NFAT transcriptional activity was monitored with two different NFATdependent reporters and was found to be higher in slow compared to fast muscles. NFAT activity is decreased by denervation in slow muscles and is increased by electrostimulation of denervated muscles with a tonic low-frequency impulse pattern, mimicking the firing pattern of slow motor neurons, but not with a phasic high-frequency pattern typical of fast motor neurons. To determine the role of NFAT, we transfected regenerating and adult rat muscles with a plasmid coding for VIVIT, a specific peptide inhibitor of Cn-mediated NFAT activation. VIVIT was found to block the expression of slow myosin heavy chain (MyHC-slow) induced by slow motor neuron activity in regenerating slow soleus muscle and to inhibit the expression of MyHC-slow transcripts and the activity of a MyHC-slow promoter in adult soleus. The role of NFAT was confirmed by the finding that a constitutively active NFATc1 mutant stimulates the MyHC-slow, inhibits the fast MyHC-2B promoter in adult fast muscles, and induces MyHC-slow expression in regenerating muscles. These results support the notion that Cn-NFAT signaling acts as a nerve activity sensor in skeletal muscle in vivo and controls nerve activity-dependent myosin switching. Mammalian skeletal muscle fibers comprise four major fiber types, including slow or type 1 and three subtypes of fast or type 2 fibers, types 2A, 2X, and 2B. Each fiber type is defined by the presence of a specific isoform of myosin heavy chain (MyHC) and by a distinct program of gene expression (1). Type 2 fibers comprise a wide spectrum of fibers with variable physiological and metabolic properties: at one extreme 2A fibers are characterized by oxidative metabolism, lowest speed of shortening, and highest resistance to fatigue, thus are more similar to type 1 fibers; at the other extreme, 2B fibers are characterized by glycolytic metabolism, highest speed, and lowest resistance to fatigue, with 2X fibers falling between these extremes. Fiber type specification is in part dictated by an early diversification of myoblast lineages during embryonic development and is subsequently modulated by neural and hormonal influences. The motor neuron firing pattern is a major determinant of the muscle fiber phenotype, and the effect of motor neuron activity can be reproduced by direct electrostimulation of denervated muscles with specific impulse patterns (2).Different signaling pathways, including calcineurin (Cn) and Ras-ERK, have been implicated in fiber type specification induced by nerve activity (3, 4). Cn, a Ca 2ϩ ͞calmodulin-regulated serine͞threonine phosphatase, acts on the four ...

In this study the stress protein response to unaccustomed maximal eccentric exercise in humans was investigated. Eleven healthy males performed 300 maximal eccentric actions with the quadriceps muscle. Biopsies from vastus lateralis were collected at 30 min and 4, 8, 24, 96, and 168 h after exercise. Cellular regulation and localization of heat shock protein (HSP) 27, αB-crystallin, and HSP70 were analyzed by immunohistochemistry, ELISA technique, and Western blotting. Additionally, mRNA levels of HSP27, αB-crystallin, and HSP70 were quantified by Northern blotting. After exercise (30 min), 81 ± 8% of the myofibers showed strong HSP27 staining ( P < 0.01) that gradually decreased during the following week. αB-Crystallin mimicked the changes observed in HSP27. After exercise (30 min), the ELISA analysis showed a 49 ± 13% reduction of the HSP27 level in the cytosolic fraction ( P < 0.01), whereas Western blotting revealed a 15-fold increase of the HSP27 level in the myofibrillar fraction ( P < 0.01). The cytosolic HSP70 level increased to 203 ± 37% of the control level 24 h after exercise ( P < 0.05). After 4 days, myofibrillar-bound HSP70 had increased ∼10-fold ( P < 0.01) and was accompanied by strong staining on cross sections. mRNA levels of HSP27, αB-crystallin, and HSP70 were all elevated the first day after exercise ( P < 0.01); HSP70 mRNA showed the largest increase (20-fold at 8 h). HSP27 and αB-crystallin seemed to respond immediately to maximal eccentric exercise by binding to cytoskeletal/myofibrillar proteins, probably to function as stabilizers of disrupted myofibrillar structures. Later, mRNA and total HSP protein levels, especially HSP70, increased, indicating that HSPs play a role in skeletal muscle recovery and remodeling/adaptation processes to high-force exercise.