NFAT is a nerve activity sensor in skeletal muscle and controls activity-dependent myosin switching

Abstract

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 ...

Activity-dependent repression of muscle genes by NFAT

“…Swoap et al (37) concluded from in vitro and in vivo studies that CaN and NFAT are not sufficient to dictate the slow muscle program, consistent with our previous analysis of the TnI SURE (15,27). On the other hand, McCullagh et al (36) showed that transcription from an artificially trimerized IL-2 NFAT site is selectively enhanced by slow-patterned activity, a finding we have reproduced here (Fig. 6A).…”

“…6A). Consistent with previous reports (36,37), expression of the 3xNFAT construct was Ϸ3-fold higher in the soleus than EDL muscles (soleus, 272% Ϯ 23% vs. EDL, 100% Ϯ 25%). NFATc1 knockdown significantly reduced transcription of the 3xNFAT reporter in the soleus (soleus, 272% Ϯ 23% vs. soleus ϩ NFATc1 siRNAs, 117% Ϯ 11%, P Ͻ 0.05) but had no effect in transfected EDL muscle (EDL, 100% Ϯ 25% vs. EDL ϩ NFATc1 siRNAs, 93% Ϯ 21%).…”

“…6A). VIVIT antagonized slow stimulationmediated increases in MHC-I promoter activity, but it was unclear whether it did so by affecting direct binding of NFAT to the promoter (36). There is also evidence that the CaN/NFAT pathway may require interaction with other signaling pathways and transcription factors to regulate slow muscle genes (38,39,43).…”

“…6). To confirm that NFAT activity is higher in the soleus than in EDL muscle (36), and that the siRNA effectively knocks down NFATc1, muscles were cotransfected with a luciferase reporter construct driven by trimerized IL-2 NFAT sites (3xNFAT), and with either NFATc1 siRNAs or nontargeting control siRNAs (Fig. 6A).…”

PGC-1α regulates a HIF2α-dependent switch in skeletal muscle fiber types

“…Calmodulin activation through binding to calcium promotes an increase in calcinueurin A activity, which has been shown to result in the exclusion of NFAT from the nucleus, to subsequently increase the activity of MEF2 and drive a slow-type fiber switch. PGC-1α has also been shown to coactivate MEF2 to promote a transition toward oxidative fiber types, a process that is thus likely to be potentiated through the PGC-1α-dependent induction of HIF2α (41)(42)(43)(44)(45)(46)(47).…”