Calcineurin-NFAT signaling and neurotrophins control transformation of myosin heavy chain isoforms in rat soleus muscle in response to aerobic treadmill training.

Author:Liu, Wenfeng
Position:Research article - Report


Contraction of the skeletal muscle requires the function of myosin heavy chain (MyHC). Isoforms of MyHC perform a variety of pleiotropic functions, depending on the isozyme type, the content, composition and distribution of skeletal muscle fiber, and the expression of neural, hormonal and mechanical factors. Skeletal muscles can de velop into two types of fibers: fast and slow, that differ in the pattern of expression of MyHC isoforms. Several hypotheses have been proposed to explain the mechanism of action of skeletal muscle fibers. A variety of different signaling pathways and molecular mechanisms, including calcineurin (CaN)/nuclear factor of activated t-cells (NFAT) signaling, [Ca.sup.2+]/calmodulin-dependent kinase (CaMK) signaling, histone deacetylase(HDAC)/ myocyte enhancer factor 2 (MEF2) signaling, myogenic regulatory factor (MRFS) pathway, Ras/MAPK signaling, Myostatin and Wnt signaling, and PGC-l[alpha]/[beta], AMPK and PPAR5 signaling have been shown to regulate gene expression and the choice of MyHC isoforms in the skeletal muscle (Carlsen et al., 2000). CaN-NFAT signaling is also involved in T cell differentiation and maturation, production of cytokines, vascular smooth muscle cell proliferation, synaptic transmission and myocardial hypertrophy. More importantly, some studies have demonstrated its role in the transformation of skeletal muscle fibers (Dupont-Versteegden et al., 2002; Serfling et al., 2006). Calcineurin is a cyclosporine- sensitive, calcium-regulated serine/threonine phosphatase, shown to be essential for skeletal muscle remodeling, where it facilitates the transduction of extracellular signals to the nucleus by targeting members of the NFAT family of transcription factors (Bassel-Duby et al., 2006). McCullagh et al. (2004) showed that expression of a constitutively active form of NFAT (NFATc1) stimulates expression of the MyHCslow isoforms in regenerating muscles, and inhibits the fast MyHC IIB promoter in fast muscles of the adult. These results support the hypothesis that CaN-NFAT signaling acts as a sensor of nerve activity in skeletal muscles in vivo and consequently controls nerve activity-dependent switch in expression of MyHC isoforms. NFATc1, NFATc2 and NFATc3 have been detected in both the cytoplasm and nucleus of skeletal muscle cells. Despite the above findings on the role of NFAT signaling in the specification of skeletal muscle fiber type, the issue --remains controversial (Schiaffino et al., 2002). In one study, Bigard et al. (2000) showed that CaN-mediated synergistic activation of NFAT (by phosphorylation) leads to the expression of slow muscle fiber-associated proteins in cooperation with the myocyte enhancer factor, and contributes to the specification of skeletal muscle fiber type. However, the involvement of CaN-NFAT signaling in exercise training-induced skeletal myofiber transformation is unclear.

Although NFAT is a key transcriptional regulator of neuronal development and function, it is poorly characterized as a possible downstream target of nerve growth factor (NGF) in neurons. Groth et al. (2007) showed that NGF can induce NFAT-dependent transcription of brain-derived neurotrophic factor (BDNF) and COX-2 in dorsal root ganglion cells. Nguyen et al. (2009) reported that NGF can repress the expression of GAP-43 (growth associated protein 43) in PC12 cells and in cultured cortical neurons through signaling mediated by NFAT3. Finally, Stefos et al. (2013) provided evidence supporting the hypothesis that CaN-NFAT signaling mediates gene regulatory effects of NGF in neurons.

An increasing quantity of experimental data supports the hypothesis that mature adult mammalian and human skeletal muscles maintain a high degree of plasticity, even after development is complete. The expression of skeletal muscle fiber type-specific proteins and changes in the composition of muscle fiber depend on developmental factors, neuromuscular activity, muscle load, hormonal levels and aging (Pette and Staron, 1997). Previous studies have reported that exercise can affect the composition and distribution of muscle fiber types. For example, increased neuromuscular activity results in a shift in MyHC isoform from fast to slow muscle fiber (Pette and Staron, 1997), while inactivity leads to a general shift in MyHC expression and associated metabolic properties along the following line of progression- from type I [right arrow] IIA [right arrow] IIX [right arrow] IIB (Talmadge., 2000). It is well documented that exercise induces several physiological and biochemical changes in the brain. Different mechanisms, including CaN and Ras-ERK signaling, have been implicated in fiber type specification induced by nerve activity (Murgia et al., 2000; Sharma et al., 1991). Wang et al. (2002a; 2002b) found that regionalization during re-innervation of muscle fiber type is indirect evidence of the important role played by muscle fiber composition during normal innervation. In cases of re-innervation of the lower limb muscles in rats by the sciatic nerve, and re-innervation of the gastrocnemius muscle in rats by its own nerve, quantitative analysis of the distribution of muscle fiber type indicated that the normal muscle fiber type has a "mosaic" distribution, with a significantly similar fiber aggregation style, called fiber type grouping. The detection of similar fiber aggregation suggests that at least part of the shift in muscle fiber type would allow innervation by different types of motor neurons (Wang et al., 2002a; 2002b).

Neurons influence muscle fiber type-specific protein expression because changes in nerve activity can induce muscle fiber to release neurotrophic factors such as neuregulin even two species passing through the joint action. Mousavi et al. (2004) investigated the role of ciliary neurotrophic factor (CNTF) and brain-derived neurotrophic factor (BDNF) in the survival and maturation of a subset of motor neurons innervating the extensor digitorum longus (EDL) and tibialis anterior (TA) muscles. Their findings demonstrated the importance of muscle-derived BDNF in the survival and maturation of a subpopulation of motor neurons, and the significance of MyHC IIB muscle fibers during neonatal development of the neuromuscular junction. Carrasco et al. (2003) found that during normal postnatal development in rats, the expression of neurotrophin (NT)-4/5 in the slow-twitch soleus muscles is indispensable to the fast to slow conversion of MyHC isoforms. Simon et al. (2003) found that the expression of NT-4 in neurons following denervation or reinnervation can selectively promote the recovery of the slow motor units. In vitro, Rende et al. (2000) showed that another neurotrophic factor NGF regulates myoblast proliferation and differentiation by signaling through its specific receptor Tyrosine Kinase A (TrK A). Despite the above findings, the expression and distribution of different types of neurotrophic factors, and their respective roles in determining the type of muscle fiber needs to be explored further. NGF, BDNF and NT-3 are three important members of the family of neurotrophic factors, mainly expressed in the brain and peripheral tissues, and known to affect neuronal survival and differentiation. Interestingly, neurotrophic factors are expressed not only by neurons, but also by muscle cells. However, it is not known whether their expression is specific to the type of muscle fiber, or if specific stimulation conditions such as long-term exercise training can change their expression in the muscle.

In adult vertebrates, subtypes of skeletal myofibers differ markedly in their contractile physiology, metabolic capabilities, ultrastructural morphology, and susceptibility to fatigue. In vivo experiments need to be conducted in order to establish the contribution of the signaling pathways and molecular mechanisms discussed above to pathophysiological alterations in MyHC isoforms. In this study, we investigated the activity of CaN-NFAT signaling, and changes in the expression and content of NGF, BDNF and NT-3 in response to aerobic treadmill training. Our results suggest that NGF, BDNF and NT-3 regulate gene expression in myocytes of the soleus muscle and neurons of the striatum by regulating the activity of the calcineurin-NFAT signaling pathway.


Ethics statement

All animal procedures were approved by the local ethics committee (the Institutional Review Board of Hunan Normal University) and the Guidelines for Care and Use of Laboratory Animals (2011). Disposal of animals was done in accordance with "The guidance on the care of laboratory animals'" (The provisions were issued in 2006 by the Ministry of Science and Technology of the People's Republic of China).

Experimental animals and treatments

Experimental animals: Specific pathogen-free 2-month-old male Sprague-Dawley (SD) rats (n=24, 220 [+ or -] 10 g) were supplied by the Animal Center of East Biotechnology Services Company (Changsha, Hunan, China; License number: Xiang scxk 2009-003). Four rats were housed in one standard cage with free access to food and water. All animals were kept in an air-conditioned room maintained at a constant temperature of 20[degrees]C to 25[degrees]C with a relative humidity of 45%-55%. Rats were subjected to a cycle of 12 h light and 12 h darkness. All animals were acclimated to laboratory conditions for 1 week, prior to the start of the experiment. A total of 24 rats were randomly assigned to three groups--sedentary control group (Con, n = 8), moderate-intensity aerobic exercise runner group (M-Ex, n = 8), and high-intensity aerobic exercise runner group (H-Ex, n = 8).

Exercise protocol: The aerobic exercise regime used in this study was proposed by Shuzhe et al. (2008), with reference to the exercise load standards of Bedford et al. (1979).All animals in the M-Ex and H-Ex groups were first subjected to a 5-day adaptation period on a rat treadmill (slope gradient 0%...

To continue reading