Different patterns of motor nerve activity drive distinctive programs of gene transcription in skeletal muscles, thereby establishing a high degree of metabolic and physiological specialization among myo®ber subtypes. Recently, we proposed that the in¯uence of motor nerve activity on skeletal muscle ®ber type is transduced to the relevant genes by calcineurin, which controls the functional activity of NFAT (nuclear family of activated T cell) proteins. Here we demonstrate that calcineurin-dependent gene regulation in skeletal myocytes is mediated also by MEF2 transcription factors, and is integrated with additional calcium-regulated signaling inputs, speci®cally calmodulin-dependent protein kinase activity. In skeletal muscles of transgenic mice, both NFAT and MEF2 binding sites are necessary for properly regulated function of a slow ®ber-speci®c enhancer, and either forced expression of activated calcineurin or motor nerve stimulation up-regulates a MEF2-dependent reporter gene. These results provide new insights into the molecular mechanisms by which specialized characteristics of skeletal myo®ber subtypes are established and maintained.

Molecular signaling pathways linking increases in skeletal muscle usage to alterations in muscle size have not been identified. In the present study, we tested the hypothesis that calcineurin, a calcium-regulated phosphatase recently implicated in the signaling of some forms of cardiomyopathic growth, is required to induce skeletal muscle hypertrophy and muscle fiber type conversions associated with functional overload in vivo. Administration of the specific calcineurin inhibitors cyclosporin (CsA) or FK506 to mice, for which the fast plantaris muscle was overloaded for 1-4 weeks, prevented the rapid doubling of mass and individual fiber size and the 4 -20-fold increase in the number of slow fibers that characterize this condition. CsA treatment influenced the expression of muscle myofibrillar protein genes in a way reflective of fiber phenotype transformations but only in the long term of the overload condition, suggesting that the control of this growth response by calcineurin is not limited to the transcriptional activation of these muscle-specific genes. Clinically, these results provide insight to the post-surgical muscle wasting and weakness observed in recovering transplant recipients administered therapeutic dosages of these immunosuppressants.The amount and type of contractile proteins incorporated into the myofibrils of skeletal muscle fibers are major determinants of the size, strength, and speed of these cells (1). To date, the molecular events linking muscle usage to the cellular expression and accumulation of these proteins are unknown. Recently, calcineurin, a cytoplasmic calcium-regulated phosphatase implicated in the pathogenesis of hypertrophic cardiomyopathy (2, 3), has emerged as a possible candidate in the signaling of skeletal muscle cellular growth and the fiber type transformations (4) of these cells. Calcineurin is an enticing prospect as a regulatory enzyme in this signaling because its selective activation of NF-AT (nuclear factor of activated T cells) transcription factors in response to sustained increases in intracellular calcium concentrations (5) is reminiscent of calcium fluctuations provoked by the activation of muscle cells during extensive contractile work (4, 6).Typically, a fast weight-bearing muscle subjected to the functional loss of its synergists will compensate by displaying within 2-4 weeks a doubling of mass and individual fiber sizes and an increase in strength (7,8). A muscle overloaded in this manner will also contract more slowly as a result of rapid fiber type transformations characterized by an increase in the number of fibers exhibiting slower, more energy-efficient contractile and calcium-handling proteins (7-9). In rodent fast muscle, the myosin heavy chain (MHC) 1 enzyme component of the major contractile protein myosin displays a conversion pattern in response to overload that follows from the fastest to the slowest isoform in the order MHC IIb 3 IIx 3 IIa 3 I/slow. The signaling of these adaptations may well be mediated by calcineurin since most of the fun...

Calcineurin-dependent pathways have been implicated in the hypertrophic response of skeletal muscle to functional overload (OV) (Dunn, S.E., J.L. Burns, and R.N. Michel. 1999. J. Biol. Chem. 274:21908–21912). Here we show that skeletal muscles overexpressing an activated form of calcineurin (CnA*) exhibit a phenotype indistinguishable from wild-type counterparts under normal weightbearing conditions and respond to OV with a similar doubling in cell size and slow fiber number. These adaptations occurred despite the fact that CnA* muscles displayed threefold higher calcineurin activity and enhanced dephosphorylation of the calcineurin targets NFATc1, MEF2A, and MEF2D. Moreover, when calcineurin signaling is compromised with cyclosporin A, muscles from OV wild-type mice display a lower molecular weight form of CnA, originally detected in failing hearts, whereas CnA* muscles are spared this manifestation. We also show that OV-induced growth and type transformations are prevented in muscle fibers of transgenic mice overexpressing a peptide that inhibits calmodulin from signaling to target enzymes. Taken together, these findings provide evidence that both calcineurin and its activity-linked upstream signaling elements are crucial for muscle adaptations to OV and that, unless significantly compromised, endogenous levels of this enzyme can accommodate large fluctuations in upstream calcium-dependent signaling events.

Utrophin levels have recently been shown to be more abundant in slow vs. fast muscles, but the nature of the molecular events underlying this difference remains to be fully elucidated. Here, we determined whether this difference is due to the expression of utrophin A or B, and examined whether transcriptional regulatory mechanisms are also involved. Immunofluorescence experiments revealed that slower fibers contain significantly more utrophin A in extrasynaptic regions as compared with fast fibers. Single-fiber RT-PCR analysis demonstrated that expression of utrophin A transcripts correlates with the oxidative capacity of muscle fibers, with cells expressing myosin heavy chain I and IIa demonstrating the highest levels. Functional muscle overload, which stimulates expression of a slower, more oxidative phenotype, induced a significant increase in utrophin A mRNA levels. Because calcineurin has been implicated in controlling this slower, high oxidative myofiber program, we examined expression of utrophin A transcripts in muscles having altered calcineurin activity. Calcineurin inhibition resulted in an 80% decrease in utrophin A mRNA levels. Conversely, muscles from transgenic mice expressing an active form of calcineurin displayed higher levels of utrophin A transcripts. Electrophoretic mobility shift and supershift assays revealed the presence of a nuclear factor of activated T cells (NFAT) binding site in the utrophin A promoter. Transfection and direct gene transfer studies showed that active forms of calcineurin or nuclear NFATc1 transactivate the utrophin A promoter. Together, these results indicate that expression of utrophin A is related to the oxidative capacity of muscle fibers, and implicate calcineurin and its effector NFAT in this mechanism.