Aging skeletal muscles suffer a steady decline in mass and functional performance, and compromised muscle integrity as fibrotic invasions replace contractile tissue, accompanied by a characteristic loss in the fastest, most powerful muscle fibers. The same programmed deficits in muscle structure and function are found in numerous neurodegenerative syndromes and disease-related cachexia. We have generated a model of persistent, functional myocyte hypertrophy using a tissue-restricted transgene encoding a locally acting isoform of insulin-like growth factor-1 that is expressed in skeletal muscle (mIgf-1). Transgenic embryos developed normally, and postnatal increases in muscle mass and strength were not accompanied by the additional pathological changes seen in other Igf-1 transgenic models. Expression of GATA-2, a transcription factor normally undetected in skeletal muscle, marked hypertrophic myocytes that escaped age-related muscle atrophy and retained the proliferative response to muscle injury characteristic of younger animals. The preservation of muscle architecture and age-independent regenerative capacity through localized mIgf-1 transgene expression suggests clinical strategies for the treatment of age or disease-related muscle frailty.
Localized synthesis of insulin-like growth factors (IGFs) has been broadly implicated in skeletal muscle growth, hypertrophy and regeneration. Virally delivered IGF-1 genes induce local skeletal muscle hypertrophy and attenuate age-related skeletal muscle atrophy, restoring and improving muscle mass and strength in mice. Here we show that the molecular pathways underlying the hypertrophic action of IGF-1 in skeletal muscle are similar to those responsible for cardiac hypertrophy. Transfected IGF-1 gene expression in postmitotic skeletal myocytes activates calcineurin-mediated calcium signalling by inducing calcineurin transcripts and nuclear localization of calcineurin protein. Expression of activated calcineurin mimics the effects of IGF-1, whereas expression of a dominant-negative calcineurin mutant or addition of cyclosporin, a calcineurin inhibitor, represses myocyte differentiation and hypertrophy. Either IGF-1 or activated calcineurin induces expression of the transcription factor GATA-2, which accumulates in a subset of myocyte nuclei, where it associates with calcineurin and a specific dephosphorylated isoform of the transcription factor NF-ATc1. Thus, IGF-1 induces calcineurin-mediated signalling and activation of GATA-2, a marker of skeletal muscle hypertrophy, which cooperates with selected NF-ATc isoforms to activate gene expression programs.
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 ...
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