Muscle is one of the four major types of tissue in animals. It is a soft tissue primarily responsible to create movement by the transformation from chemical (ATP) to mechanical energy. It is characterized by the following functions: contractile move and gravitational stability of body matters during circulation, defecation, respiration, urination, digestion, and childbirth (Purves et al., 2004; Carpenter, 2007). There are three histological types of muscle—cardiac, smooth and skeletal—which differ in appearance, physiology, and function. The skeletal muscle type is of special interest of this thesis and will be further studied in the following sections.
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Skeletal muscles are, as the name implies, bound to the skeleton by means of tendons, and they are described as striated and voluntary. Fundamentally, they consist of long and cylindrical cells called muscle fibers, which are actually huge single cells that form during development by the fusion of many separate cells, called myoblasts. Each cell contains multiple nuclei adjacent to the plasma membrane and are around 10-100m in diameter and 30 cm long. The bulk of the cytoplasm inside myofibers is made up of myofibrils, which is the name given to the basic contractile elements of the muscle cell. Inside each myofibril are thin actin filaments and thick specific muscle isoforms of myosin II filaments that participate throughout contraction (Alberts et al., 2008). In the majority of areas of the myofibrils, six actin filaments enclose each myosin filament, and inversely, each actin filament lies inside a triangle of three myosin filaments. The myofibril entails repeating units (sarcomeres), which are the muscular contraction units, of 2.2 microns in length approximately. Cardiac muscle and smooth muscle also contain sarcomeres, although the organization is not as regular as that in skeletal muscle (Alberts et al., 2008; Purves et al., 2004).
Additionally, connective tissue builds the space for nerves and blood capillaries to get in contact with each cell (Saladin, 2003).
Classes of muscle fibers
Muscle fibers can vary concerning metabolic processes or performance. In fact, some fibers act relatively slowly but are resilient to fatigue, whereas others react and fatigue more rapidly. Certainly, skeletal muscles can be grouped as fast and slow twitch fibers and also by their different expressions of myosin heavy chain (MHC) isoforms as follows:
- Type I (MHC-I): Also called slow oxidative (SO) or slow-twitch. These fibers are abundant in myoglobin, blood capillaries and mitochondrial activity. These fibers do not fatigue easily and are more adjusted to aerobic respiration, hence not generating lactic acid.
- Type II (MHC-II): Also called fast glycolytic (FG) or fast-twitch. Their metabolism is more adapted for the phosphagen and glycogen–lactic acid systems. Their fast contractions results from a capable sarcoplasmic reticulum that processes Ca2 rapidly. However, they fatigue more easily than because of higher amounts of lactic acid production.
The existence of a subgroup of fibers has been recognized by many authors. They have been identified only in the fast-twitch fibers: types IIA, IIB and IIX. Type IIA combine fast-twitch reactions with fatigue-resistant aerobic metabolism (type I-like), while type IIB are the common type known. Remarkably, human skeletal muscle does not usually contain MHC-IIB, except from some endurance-trained athletes (Spangenburg and Booth, 2003; Zierath & Hawley, 2004; Schiaffino and Reggiani, 1994; Smerdu et al., 1994; Saladin, 2003). Finally, fibers showing two MHC isoforms (the hybrid types: I/IIA, IIAX, IIXB) can additionally be found in rodent muscle (Schiaffino and Reggiani, 1994; Staron and Pette, 1993).
Immunohistochemical procedures with antibodies against the specific MHC isoforms can be used for histological studies (Schiaffino et al., 1989; Lucas et al., 2000). Lastly, it is important to note that not all muscles are composed of both SO and FG fibers, but the proportions of these fiber types differ from one muscle to another (Bloember & Quadrilatero, 2012).
Vertebrate skeletal myogenic differentiation proceeds through three stages: determination of the muscle progenitor cells, called satellite cells, into myoblasts; myoblasts proliferation and migration; and lastly, fusing to form multinucleated myotubes to produce mature muscle:
Muscle fibersare made from the fusion ofmyoblastsinto myotubes, a fiber formed from multi-nucleated cells. In the early development of an embryo, after progenitor cells myogenic lineage commitment, newly formed myoblasts will proliferate if sufficient fibroblast growth factor(FGF) signal is available, without differentiating. When these factors are depleted, the myoblasts stop division and produce fibronectinto the extracellular matrix binding to it by the integrin molecules, their main fibronectin receptor (Menko & Boettiger, 1987; Sastry et al., 1992; Rosen et al., 1991). The second phase encompasses the alignment of the myoblasts together into series and then into myotubes. This stage is regulated by cell membrane glycoproteins, involving different cadherins and CAMs (Knudsen et al., 1990; Donalies et al., 1991; Mege et al., 1992; Peck & Walsh, 1993). The last and third stage is when cells are fused. In this phase,calciumions are essential for development (Shainberg et al., 1969; David et al., 1981). This fusion is controlled by meltrins (e.g., c-Met), a group of metalloproteinases.Many other myogenic enzymes and transcription factors have been reported that contribute in this process (Wei et al., 1998; Vlahopoulos et al., 2005).
The extracellular signals that induce differentiation of muscle progenitor cells (satellite cells) to turn into myoblasts are present only transitorily, determining the differentiation stage of these cells. Subsequently, these signals trigger production of intracellular factors that uphold the myogenic program although the inducing signals are gone. The identification and functions of some of these myogenic proteins are discussed in the next sections.
Nestin in muscle
In vivo, nestin is expressed during muscle development in somites and briefly during differentiation of skeletal muscle (Kachinsky et al., 1994; Sejersen and Lendahl, 1993). Nestin expression similarly occurs during the development of cardiac muscle (Kachinsky et al., 1995). In proliferating myoblasts, nestin is weakly present, but it increases in a precise pattern when the differentiation process starts. After about 48 hours of differentiation the expression reaches its peak, and at this time, several myogenic markers are already detected. This IF appears co-expressed with desmin at the Z-plate (Sejersen and Lendahl, 1993). Subsequently, upon further differentiation, nestin is down-regulated and finally disappears in the fully differentiated muscle. As an exception, it is to be noted that specific expression remains high in neuromuscular synapses (NMJs) and in myotendous junctions (MTJs, Vaittinen et al., 1999). Expression of nestin rises in muscle when there is regeneration process upon damage, and satellite cells begin to proliferate and form myoblasts, which can differentiate and fuse together existing muscle fibers (Vaittinen et al., 2001). The process of regeneration is similar to normal muscle development and differentiation.
Kenneth Day et al. (2007) reported a correlation of nestin expression and skeletal muscle myogenesis, more specifically, as a marker of the quiescent state satellite cells. With this, they contributed with new insights about the molecular signature of these progenitor cells and a pioneering method to isolate and characterize them regardless of their origin or embryonic fiber type composition. In this work, the study of nestin expression contributed with the understanding of the dynamics reflected in the cycle of satellite cell self-renewal.
As discussed before, IFs can regulate cellular signaling cascades by interactions with various signaling molecules, such as receptors and kinases. An essential way to regulate cellular interactions between IFs and other proteins is by phosphorylation. This phosphorylation may also affect the polymerization process on IFs or regulate receptors and kinases’ pathways (Hyder et al., 2008.) In the case of nestin, its long C-terminus could perform as a signaling linker between the different cytoskeletal components and other cells. It is also important to understand that nestin’s role could not only be passive since it can also participate in cellular signaling as a supportive structure for signaling molecules.
Cdk5 and p35
Nestin can act as a molecular scaffold, thereby enabling the activation and deactivation of kinases and linked proteins. This is the case of the cyclin-dependent kinase 5 (Cdk5) signaling pathways in muscle progenitor cells (Hyder et al., 2008; Sahlgren et al., 2003).
Cdk5 is a unique member of the CDK family, as it is not a checkpoint kinase for cell cycle progression control but a regulator of further post-mitotic pathways. Around 20 functionally different proteins have been related to act as Cdk5´s substrates concerning cell adhesion, membrane trafficking, transport and cytoskeleton dynamics (Wang et al., 2005). Similarly, Cdk5 is a kinase involved in the neuronal migration during development (Hirasawa et al., 2004), regulating the protein composition on neuromuscular synapses (Fu et al., 2001) and controlling apoptosis (Ahuja et al., 1998).[IM1] [IM2] Cdk5’s expression is diverse and interesting. Specifically, is present in Leydig cells, Sertoli cells, in insulin-secreting pancreatic cells, muscle progenitor cells and in NMJs (Lazaro et al., 1997; Philpott et al., 1997; Fu et al., 2001, 2005; Musa et al., 1998; Lin et al., 2009). Remarkably, Cdk5 seems to express in the same tissues as nestin.
Cdk5 is activated by the neuronal regulatory subunits p35 and its cleaved form p25, or alternatively by p39 (Lew et al., 1994; Ko et al., 2001). Recruitment of Cdk5 to the membrane occurs through its interaction with a myristoylated p35. p35 is a short-lived protein, as the kinase can regulate its own activity by phosphorylation of p35 resulting in the proteasomal degradation of p35 and the inhibition of the kinase activity (Patrick et al., 1998). p35 can be also deregulated by calpain1-mediated cleavage to the 25kDa C-terminal fragment of the activator, p25 (reviewed by Dhavan and Tsai, 2001). The myristoylation serves as a biochemical signature that locates both p35 and the kinase principally to the cell membrane. This sequence is absent in p25. Moreover, p35’s half-life is two to three times shorter compared to p25, making p25 a more stable molecule that produces persistent abnormal and unwanted Cdk5 activity. Consequently, a hyperactive p25–Cdk5 complex translocates to the nucleus, and triggers cell death in neurodegenerative diseases. Alzheimer’s disease is one significant consequence of the negative effects produced by p25 accumulation in neurons (Patrick et al., 1999).
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Cdk5 has been reported to play a crucial role on myoblasts performance. Studies indicate that during differentiation, the protein’s expression is activated while it localizes to the nucleus. Moreover, overexpression studies show that Cdk5 promotes myogenesis (Lazaro et al., 1997). And on the other hand, inhibition of myogenesis has been described when deficiencies in Cdk5 activity are present (Lazaroet al.,1997; Philpottet al.,1997).Remarkably, a greater interaction between Cdk5 and p35 has been observed when nestin is not copolymerized with vimentin (Eliasson et al., 1999). Furthermore, in 2003 it was reported that Cdk5 could phosphorylate nestin at threonine 316 during mitosis, keeping it soluble and protecting it from degradation. This event can occur on neurons and during muscle differentiation (Sahlgren et al., 2003).
Nestin as a determinant for muscle cell fate
Later on, Pallari et al. (2011) reported that in nestin-depleted myoblasts, as the physiological regulation of Cdk5/p35 by nestin is not present, an uncontrolled p35 processing and an abnormal Cdk5 activity during the differentiation process was observed. Moreover, they showed that nestin seems to represent a dynamic scaffold that regulates the processing of p35/Cdk5 complex within interaction and keeping it at the cell membrane. Hence, by the sequestration of Cdk5/p35, nestin appears capable to weaken p35 processing into p25 and determining myogenesis. Additionally, it is shown a bidirectional relationship between nestin and Cdk5, where this kinase regulates the structural organization and stability of the nestin that in turn is its own scaffold, thus regulating its own activity.
Nestin and NMJs
The neuromuscular junctions (NMJs) are basically the synapse that occurs between motor neurons and muscle. In mammals, presynaptic motor neurons interact with the plasma membrane of muscle fibers (sarcolemma) by the liberation of the neurotransmitter acetylcholine (ACh). In parallel, the sarcolemma has nicotinic acetylcholine receptors (nAChRs) that also serve as ligands gated ion channels. After ACh binding, these receptors depolarize muscle fibers and eventually cause muscle contraction (Nicholls et al., 2012).
Although nestin filaments are down-regulated and replaced by the IF protein desmin in fully differentiated myotubes, its expression remains high in neuromuscular synapses (NMJs) and in myotendous junctions (MTJs, Vaittinen et al., 1999). Thus, nestin has been proposed as a strengthening structure and also may function as an organizing network for acetylcholine receptors in the neuromuscular synapses (Vaittinen et al., 1999).
Likewise, it is quite clear that nestin and Cdk5 can carefully interplay in several but specific contexts. At the neuromuscular junctions (NMJs) environment, it has been described that p35, Cdk5 and nestin are enriched in the synapses in the muscle of the adult rodents (Fu et al., 2001; Vaittinen et al., 1999). Likewise, Cdk5 activity seems to be essential for the transcription of acetylcholine receptors induced by neuregulin; additionally, deficiencies in Cdk5 activity have been shown to compromise signal transduction (Wei et al., 2005; Fuet al.,2001,2005).Consequently, nestin and Cdk5/p35 have been proposed to cooperate in the organization of ErbB neuregulin receptors governing acetylcholine receptors transcription and contributing in the maintainance of the neuromuscular junction (Sahlgren et al., 2003; Schmidt et al., 2011).
Satellite cells have arisen the interest of investigators as a practical model for postnatal stem cells. Activation, proliferation, differentiation and self-renewal in vivo and in vitro experiments have been developed in murine satellite cells owing to the accessibility of markers and transgenic techniques (reviewed by Boldrin et al., 2010).
Most studies have focused in rodents’ skeletal muscle, mainly in mice. Certainly, this model has been very useful due to the difficulties existing when analyzing human skeletal muscle. More specifically, those related to the deficiency of molecular markers to discriminate satellite cells from myoblasts and other cells along with the biopsy practical complications. Nonetheless, whether the same mechanisms apply to the human remains to be demonstrated (reviewed by Boldrin et al., 2010).
Most of mouse satellite cells can be recognized by their expression of several transcription factors, enzymes and cytoskeletal structures. Among them, the paired box (Pax) transcription factors, which regulate proliferation and myogenic regulatory factors. The majority of satellite cells express Pax7 in mice and human. This molecule is a quiescent and activated cell marker. It has antibody available and it localizes in the nucleus (Seale et al,. 2000; Bosnakovski et al., 2008). Likewise, Pax3 is alternative Pax transcription factor. It is a marker for a subset of quiescent satellite cells and it has been mainly seen in transgenic Pax3-GFP+ mice (Relaix et al., 2006). Cells expressing Pax7 is believed to be important for the regeneration of myoblasts. Also the expression of Pax3 has negative effect on the differentiation and it has been shown that this transcription factor is down-regulated in muscle by ubiquitination and proteasomal degradation before differentiation can proceed. Pax transcription factors are downregulated upon further differentiation and other proteins predominate (Boutet et al., 2007).
Additionally, a marker for the activated state of satellite cells exclusively is MyoD, which belongs to the group of basic helix-loop-helix transcription factors for myogenic regulation, and has been found to be essential for myogenic determination (Sabourin et al., 1999). It is defined as a marker for activated satellite and myogenic cells in general, it is available as a mice antibody that locales in the nucleus. (Yablonka-Reuveni et al., 1999; Zammit et al,. 2004). Others markers include CD34, M-cadherin, caveolin and calcitonin for the quiescent and activated satellite cells. As seen, nestin seems to be the only quiescent marker for satellite cells available (Day et al., 2007).
Nestin knockout mouse model
In order to elucidate the physiological functions of the nestin IF in vivo, a nestin knockout (Nes-/-) mouse have been generated via gene targeting (Mohseni et al., 2011). Their results indicate that albeit Nes-/- mice are fertile and look normal, some differences from the WT mouse type have been found. For example, the amount of AChR clusters within the NMJ region is significantly higher in the diafragm tissue along with a more dispersed distribution. Furthermore, the authors suggest that nestin and the Cdk5 signaling pathway are involved during these processes. More studies on other tissues and at different environments should be further ripen in order to understand the true significance of the nestin intermediate filament’s functionality in mammals.
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