Slow-and fast-twitch myofibers of adult skeletal muscles express unique sets of muscle-specific genes, and these distinctive programs of gene expression are controlled by variations in motor neuron activity. It is well established that, as a consequence of more frequent neural stimulation, slow fibers maintain higher levels of intracellular free calcium than fast fibers, but the mechanisms by which calcium may function as a messenger linking nerve activity to changes in gene expression in skeletal muscle have been unknown. Here, fiber-type-specific gene expression in skeletal muscles is shown to be controlled by a signaling pathway that involves calcineurin, a cyclosporin-sensitive, calcium-regulated serine/threonine phosphatase. Activation of calcineurin in skeletal myocytes selectively up-regulates slow-fiber-specific gene promoters. Conversely, inhibition of calcineurin activity by administration of cyclosporin A to intact animals promotes slow-to-fast fiber transformation. Transcriptional activation of slow-fiber-specific transcription appears to be mediated by a combinatorial mechanism involving proteins of the NFAT and MEF2 families. These results identify a molecular mechanism by which different patterns of motor nerve activity promote selective changes in gene expression to establish the specialized characteristics of slow and fast myofibers.

It is now well established that stromal interaction molecule 1 (STIM1) is the calcium sensor of endoplasmic reticulum stores required to activate store-operated calcium entry (SOC) channels at the surface of non-excitable cells. However, little is known about STIM1 in excitable cells, such as striated muscle, where the complement of calcium regulatory molecules is rather disparate from that of non-excitable cells. Here, we show that STIM1 is expressed in both myotubes and adult skeletal muscle. Myotubes lacking functional STIM1 fail to show SOC and fatigue rapidly. Moreover, mice lacking functional STIM1 die perinatally from a skeletal myopathy. In addition, STIM1 haploinsufficiency confers a contractile defect only under conditions where rapid refilling of stores would be needed. These findings provide insight into the role of STIM1 in skeletal muscle and suggest that STIM1 has a universal role as an ER/SR calcium sensor in both excitable and non-excitable cells.

Peroxisome proliferator-activated receptor ␥ co-activator 1␣ (PGC-1␣) promotes mitochondrial biogenesis and slow fiber formation in skeletal muscle. We hypothesized that activation of the p38 mitogen-activated protein kinase (MAPK) pathway in response to increased muscle activity stimulated Pgc-1␣ gene transcription as part of the mechanisms for skeletal muscle adaptation. Here we report that a single bout of voluntary running induced a transient increase of Pgc-1␣ mRNA expression in mouse plantaris muscle, concurrent with an activation of the p38 MAPK pathway. Activation of the p38 MAPK pathway in cultured C2C12 myocytes stimulated Pgc-1␣ promoter activity, which could be blocked by the specific inhibitors of p38, SB203580 and SB202190, or a dominant negative p38. Furthermore, the p38-mediated increase in Pgc-1␣ promoter activity was enhanced by increased expression of the downstream transcription factor ATF2 and completely blocked by ATF2⌬N, a dominant negative ATF2. Skeletal muscle-specific expression of a constitutively active activator of p38, MKK6E, in transgenic mice resulted in enhanced Pgc-1␣ and cytochrome oxidase IV protein expression in fast-twitch skeletal muscles. These findings suggest that contractile activity-induced activation of the p38 MAPK pathway promotes Pgc-1␣ gene expression and skeletal muscle adaptation.

Endurance exercise training promotes mitochondrial biogenesis in skeletal muscle and enhances muscle oxidative capacity, but the signaling mechanisms involved are poorly understood. To investigate this adaptive process, we generated transgenic mice that selectively express in skeletal muscle a constitutively active form of calcium/calmodulin-dependent protein kinase IV (CaMKIV*). Skeletal muscles from these mice showed augmented mitochondrial DNA replication and mitochondrial biogenesis, up-regulation of mitochondrial enzymes involved in fatty acid metabolism and electron transport, and reduced susceptibility to fatigue during repetitive contractions. CaMK induced expression of peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1), a master regulator of mitochondrial biogenesis in vivo, and activated the PGC-1 gene promoter in cultured myocytes. Thus, a calcium-regulated signaling pathway controls mitochondrial biogenesis in mammalian cells.

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.

Adult skeletal muscle fibers can be categorized into fast and slow twitch subtypes based on specialized contractile and metabolic properties and on distinctive patterns of muscle gene expression. Muscle fiber-type characteristics are dependent on the frequency of motor nerve stimulation and are thought to be controlled by calcium-dependent signaling. The calcium, calmodulindependent protein phosphatase, calcineurin, stimulates slow fiber-specific gene promoters in cultured skeletal muscle cells, and the calcineurin inhibitor, cyclosporin A, inhibits slow fiber gene expression in vivo, suggesting a key role of calcineurin in activation of the slow muscle fiber phenotype. Calcineurin has also been shown to induce hypertrophy of cardiac muscle and to mediate the hypertrophic effects of insulin-like growth factor-1 on skeletal myocytes in vitro. To determine whether activated calcineurin was sufficient to induce slow fiber gene expression and hypertrophy in adult skeletal muscle in vivo, we created transgenic mice that expressed activated calcineurin under control of the muscle creatine kinase enhancer. These mice exhibited an increase in slow muscle fibers, but no evidence for skeletal muscle hypertrophy. These results demonstrate that calcineurin activation is sufficient to induce the slow fiber gene regulatory program in vivo and suggest that additional signals are required for skeletal muscle hypertrophy.Adult skeletal muscle fibers can be generally classified as fast or slow on the basis of their contractile and metabolic properties (1, 2). Certain fast fibers exhibit a glycolytic metabolism, whereas slow twitch fibers are predominantly oxidative. These properties reflect the expression of specific sets of fast and slow contractile protein isoforms of myosin heavy and light chains, tropomyosin, and troponins, as well as myoglobin.The fiber-type characteristics of adult skeletal muscle are regulated, at least in part, by the frequency of motor nerve stimulation and resulting changes in intracellular calcium levels (3-5). Tonic low frequency stimulation, either from the motor neuron or by direct electrical stimulation of muscle fibers, induces the slow fiber phenotype, whereas denervation or lack of electrical stimulation evokes a slow-to-fast fiber conversion (reviewed in Ref. 6). It is well established that slow fibers maintain higher levels of intracellular calcium (7,8), but the signaling systems responsible for activation of the slow fiber gene program remain poorly understood.Calcineurin is a calcium-, calmodulin-dependent protein phosphatase activated by changes in intracellular calcium (reviewed in Ref. 9). The properties of calcineurin have been studied most extensively in T cells, whereupon activation in response to T cell receptor signaling, calcineurin dephosphorylates nuclear factor of activated T cells (NFAT) 1 proteins, which translocate to the nucleus and act combinatorially with other transcription factors (reviewed in Ref. 10). Calcineurin-dependent activation of NFAT is driven selectively by su...

Here we describe a small family of proteins, termed MCIP1 and MCIP2 (for myocyte-enriched calcineurin interacting protein), that are expressed most abundantly in striated muscles and that form a physical complex with calcineurin A. MCIP1 is encoded by DSCR1, a gene located in the Down syndrome critical region. Expression of the MCIP family of proteins is up-regulated during muscle differentiation, and their forced overexpression inhibits calcineurin signaling to a muscle-specific target gene in a myocyte cell background. Binding of MCIP1 to calcineurin A requires sequence motifs that resemble calcineurin interacting domains found in NFAT proteins. The inhibitory action of MCIP1 involves a direct association with the catalytic domain of calcineurin, rather than interference with the function of downstream components of the calcineurin signaling pathway. The interaction between MCIP proteins and calcineurin may modulate calcineurin-dependent pathways that control hypertrophic growth and selective programs of gene expression in striated muscles.Calcineurin is a serine/threonine protein phosphatase that plays a pivotal role in developmental and homeostatic regulation of a wide variety of cell types (1, 2). The interaction of calcineurin with transcription factors of the NFAT 1 family following activation of the T cell receptor in leukocytes provides the best characterized example of how calcineurin regulates gene expression (3). Changes in intracellular calcium promote binding of Ca 2ϩ /calmodulin to the catalytic subunit of calcineurin (CnA), thereby displacing an autoinhibitory region and allowing access of protein substrates to the catalytic domain. Dephosphorylation of NFAT by activated calcineurin promotes its translocation from the cytoplasm to the nucleus, where NFAT binds DNA cooperatively with an AP1 heterodimer to activate transcription of genes encoding cytokines such as IL-2. This basic model of NFAT activation has been shown to transduce Ca 2ϩ signals via calcineurin in many cell types and to control transcription of diverse sets of target genes unique to each cellular environment (4). In each case, NFAT acts cooperatively with other transcription factors that include proteins of the AP1 (3), cMAF (5), GATA (6 -8), or MEF2 (9 -12) families. In addition to T cell activation, cellular responses controlled by calcineurin signaling include synaptic plasticity (11,13,14) and apoptosis (15,16).Recent studies of calcineurin signaling in striated myocytes of heart and skeletal muscle have expanded the scope of important physiological and pathological events controlled by this ubiquitously expressed protein. Forced expression of a constitutively active form of calcineurin in hearts of transgenic mice promotes cardiac hypertrophy that progresses to dilated cardiomyopathy, heart failure, and death, in a manner that recapitulates features of human disease (7). Moreover, hypertrophy and heart failure in these animals, and in certain other animal models of cardiomyopathy, are prevented by administration of the calcin...

Cytochrome c released from mitochondria has been proposed to be an essential component of an apoptotic pathway responsive to DNA damage and other forms of cell stress. Murine embryos devoid of cytochrome c die in utero by midgestation, but cell lines established from early cytochrome c null embryos are viable under conditions that compensate for defective oxidative phosphorylation. As compared to cell lines established from wild-type embryos, cells lacking cytochrome c show reduced caspase-3 activation and are resistant to the proapoptotic effects of UV irradiation, serum withdrawal, or staurosporine. In contrast, cells lacking cytochrome c demonstrate increased sensitivity to cell death signals triggered by TNFalpha. These results define the role of cytochrome c in different apoptotic signaling cascades.