Giorgia Pallafacchina

A protein kinase B-dependent and rapamycin- sensitive pathway controls skeletal muscle growth but not fiber type specification

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 controls nerve activity-dependent specification of slow skeletal muscle fibers but not muscle growth

Nerve activity can induce long-lasting, transcription-dependent changes in skeletal muscle fibers and thus affect muscle growth and fiber-type specificity. Calcineurin signaling has been implicated in the transcriptional regulation of slow muscle fiber genes in culture, but the functional role of calcineurin in vivo has not been unambiguously demonstrated. Here, we report that the up-regulation of slow myosin heavy chain (MyHC) and a MyHC-slow promoter induced by slow motor neurons in regenerating rat soleus muscle is prevented by the calcineurin inhibitors cyclosporin A (CsA), FK506, and the calcineurin inhibitory protein domain from cain/cabin-1. In contrast, calcineurin inhibitors do not block the increase in fiber size induced by nerve activity in regenerating muscle. The activation of MyHC-slow induced by direct electrostimulation of denervated regenerating muscle with a continuous low frequency impulse pattern is blocked by CsA, showing that calcineurin function in muscle fibers and not in motor neurons is responsible for nerve-dependent specification of slow muscle fibers. Calcineurin is also involved in the maintenance of the slow muscle fiber gene program because in the adult soleus muscle, cain causes a switch from MyHC-slow to fast-type MyHC-2X and MyHC-2B gene expression, and the activity of the MyHC-slow promoter is inhibited by CsA and FK506.

Ras is involved in nerve-activity-dependent regulation of muscle genes

Gene expression in skeletal muscle is regulated by the firing pattern of motor neurons, but the signalling systems involved in excitation–transcription coupling are unknown. Here, using in vivo transfection in regenerating muscle, we show that constitutively active Ras and a Ras mutant that selectively activates the MAPK(ERK) pathway are able to mimic the effects of slow motor neurons on expression of myosin genes. Conversely, the effect of slow motor neurons is inhibited by a dominant-negative Ras mutant. MAPK(ERK) activity is increased by innervation and by low-frequency electrical stimulation. These results indicate that Ras–MAPK signalling is involved in promoting nerve-activity-dependent differentiation of slow muscle fibres in vivo.

An adult tissue-specific stem cell in its niche: A gene profiling analysis of in vivo quiescent and activated muscle satellite cells

The satellite cell of skeletal muscle provides a paradigm for quiescent and activated tissue stem cell states. We have carried out transcriptome analyses on satellite cells purified by flow cytometry from Pax3(GFP/+) mice. We compared samples from adult skeletal muscles where satellite cells are mainly quiescent, with samples from growing muscles or regenerating (mdx) muscles, where they are activated. Analysis of regulation that is shared by both activated states avoids other effects due to immature or pathological conditions. This in vivo profile differs from that of previously analyzed satellite cells activated after cell culture. It reveals how the satellite cell protects itself from damage and maintains quiescence, while being primed for activation on receipt of the appropriate signal. This is illustrated by manipulation of the corepressor Dach1, and by the demonstration that quiescent satellite cells are better protected from oxidative stress than those from mdx or 1-week-old muscles. The quiescent versus in vivo activated comparison also gives new insights into how the satellite cell controls its niche on the muscle fiber through cell adhesion and matrix remodeling. The latter also potentiates growth factor activity through proteoglycan modification. Dismantling the extracellular matrix is important for satellite cell activation when the expression of proteinases is up-regulated, whereas transcripts for their inhibitors are high in quiescent cells. In keeping with this, we demonstrate that metalloproteinase function is required for efficient regeneration in vivo.

Autocrine and Paracrine Angiopoietin 1/Tie-2 Signaling Promotes Muscle Satellite Cell Self-Renewal

Mechanisms governing muscle satellite cell withdrawal from cell cycle to enter into quiescence remain poorly understood. We studied the role of angiopoietin 1 (Ang1) and its receptor Tie-2 in the regulation of myogenic precursor cell (mpc) fate. In human and mouse, Tie-2 was preferentially expressed by quiescent satellite cells in vivo and reserve cells (RCs) in vitro. Ang1/Tie-2 signaling, through ERK1/2 pathway, decreased mpc proliferation and differentiation, increased the number of cells in G0, increased expression of RC-associated markers (p130, Pax7, Myf-5, M-cadherin), and downregulated expression of differentiation-associated markers. Silencing Tie-2 had opposite effects. Cells located in the satellite cell neighborhood (smooth muscle cells, fibroblasts) upregulated RC-associated markers by secreting Ang1 in vitro. In vivo, Tie-2 blockade and Ang1 overexpression increased the number of cycling and quiescent satellite cells, respectively. We propose that Ang1/Tie-2 signaling regulates mpc self-renewal by controlling the return to quiescence of a subset of satellite cells.

NFAT isoforms control activity-dependent muscle fiber type specification

The intracellular signals that convert fast and slow motor neuron activity into muscle fiber type specific transcriptional programs have only been partially defined. The calcium/calmodulin-dependent phosphatase calcineurin (Cn) has been shown to mediate the transcriptional effects of motor neuron activity, but precisely how 4 distinct muscle fiber types are composed and maintained in response to activity is largely unknown. Here, we show that 4 nuclear factor of activated T cell (NFAT) family members act coordinately downstream of Cn in the specification of muscle fiber types. We analyzed the role of NFAT family members in vivo by transient transfection in skeletal muscle using a loss-of-function approach by RNAi. Our results show that, depending on the applied activity pattern, different combinations of NFAT family members translocate to the nucleus contributing to the transcription of fiber type specific genes. We provide evidence that the transcription of slow and fast myosin heavy chain (MyHC) genes uses different combinations of NFAT family members, ranging from MyHC-slow, which uses all 4 NFAT isoforms, to MyHC-2B, which only uses NFATc4. Our data contribute to the elucidation of the mechanisms whereby activity can modulate the phenotype and performance of skeletal muscle.

The Mitochondrial Calcium Uniporter (MCU): Molecular Identity and Physiological Roles

The direct measurement of mitochondrial [Ca2+] with highly specific probes demonstrated that major swings in organellar [Ca2+] parallel the changes occurring in the cytosol and regulate processes as diverse as aerobic metabolism and cell death by necrosis and apoptosis. Despite great biological relevance, insight was limited by the complete lack of molecular understanding. The situation has changed, and new perspectives have emerged following the very recent identification of the mitochondrial Ca2+ uniporter, the channel allowing rapid Ca2+ accumulation across the inner mitochondrial membrane.

NFATc1 nucleocytoplasmic shuttling is controlled by nerve activity in skeletal muscle.

Calcineurin-NFAT signaling has been shown to control activity-dependent muscle gene regulation and induce a program of gene expression typical of slow oxidative muscle fibers. Following Ca2+-calmodulin stimulation, calcineurin dephosphorylates NFAT proteins and induces their translocation into the nucleus. However, NFAT nuclear translocation has never been investigated in skeletal muscle in vivo. To determine whether NFATc1 nucleocytoplasmic shuttling depends on muscle activity, we transfected fast and slow mouse muscles with plasmids coding for an NFATc1-GFP fusion protein. We found that NFATc1-GFP has a predominantly cytoplasmic localization in the fast tibialis anterior muscle but a predominantly nuclear localization in the slow soleus muscle, with a characteristic focal intranuclear distribution. Two hours of complete inactivity, induced by denervation or anaesthesia, cause NFATc1 export out of the nucleus in soleus muscle fibers, whereas electrostimulation of tibialis anterior with a low-frequency tonic impulse pattern, mimicking the firing pattern of slow motor neurons, causes NFATc1 nuclear translocation. The activity-dependent nuclear import and export of NFATc1 is a rapid event, as visualized directly in vivo by two-photon microscopy. The calcineurin inhibitor cain/cabin1 causes nuclear export of NFATc1 both in normal soleus and stimulated tibialis anterior muscle. These findings support the notion that in skeletal muscle NFATc1 is a calcineurin-dependent nerve activity sensor.

Role of satellite cells in muscle growth and maintenance of muscle mass.

Changes in muscle mass may result from changes in protein turnover, reflecting the balance between protein synthesis and protein degradation, and changes in cell turnover, reflecting the balance between myonuclear accretion and myonuclear loss. Myonuclear accretion, i.e. increase in the number of myonuclei within the muscle fibers, takes place via proliferation and fusion of satellite cells, myogenic stem cells associated to skeletal muscle fibers and involved in muscle regeneration. In developing muscle, satellite cells undergo extensive proliferation and most of them fuse with myofibers, thus contributing to the increase in myonuclei during early postnatal stages. A similar process is induced in adult skeletal muscle by functional overload and exercise. In contrast, satellite cells and myonuclei may undergo apoptosis during muscle atrophy, although it is debated whether myonuclear loss occurs in atrophying muscle. An increase in myofiber size can also occur by changes in protein turnover without satellite cell activation, e.g. in late phases of postnatal development or in some models of muscle hypertrophy. The relative role of protein turnover and cell turnover in muscle adaptation and in the establishment of functional muscle hypertrophy remains to be established. The identification of the signaling pathways mediating satellite cell activation may provide therapeutic targets for combating muscle wasting in a variety of pathological conditions, including cancer cachexia, renal and cardiac failure, neuromuscular diseases, as well as aging sarcopenia.

How is muscle phenotype controlled by nerve activity

Abstract Motor neurons are known to affect muscle growth and fiber type profile (fast/slow, oxidative/glycolytic) by regulating muscle gene expression. However, the mechanism by which the information contained in specific action potential patterns is decoded by the transcriptional machinery of muscle fiber nuclei remains to be established. This is a basic issue in nerve/muscle biology, which has major implications in neurology, sport medicine and aging. We describe here a general strategy aimed at identifying the signal transduction pathways mediating the effects of nerve activity. This approach is based on the overexpression of constitutively active or dominant negative transduction factors in regenerating skeletal muscle.