Skeletal muscle contraction is under the tight control of motor nerve activity by means of a unique muscle and nerve common structure named the “neuromuscular synapse” or “neuromuscular junction” (Fig. 3.1).

The NMJ or motor endplate could be functionally and anatomically divided into four distinct regions: a presynaptic, a synaptic (cleft), a postsynaptic region, and a subsynaptic microdomain.

There is a large body of evidence suggesting that skeletal muscles, and in particular lower limb and antigravity postural muscles, undergo several adaptive processes during prolonged inactivity or unloading during exposure to microgravity (μG) mostly characterized by structural and metabolic changes, which thereafter might be largely responsible for the altered skeletal muscle structure and function. For instance, the μG-induced muscle atrophy is well known since the beginning of the human space program, as well as the related reduction in motor performance in astronauts, which is the likely one cause for the decrement in skeletal muscle mass and strength and in resistance to fatigue, severely affecting both “inflight” and “extravehicular” crew members’ mission duties.

Needless to say that the beneficial effects of exercise for the musculoskeletal quality and neuromuscular control are well accepted today. However, studies on mature NMJs of normal adult skeletal muscle showed continuous remodeling mechanisms even under physiological conditions suggesting a more or less continuum microstructural representation in normal healthy skeletal muscle depending on everyday moderate activities and fitness, throughout human life performances, and even chaperoning healthy aging on Earth that need to be considered for investigations. For instance, extended periods of increased or decreased neuromuscular activity more extensively stimulate remodeling, including changes in the overall NMJ microstructure and size. Recently, the role of exercise in maintaining the integrity of the NMJ at molecular level has been extensively reviewed (Nishimune et al. 2014). Overall, the results suggest that NMJ hypertrophy and the increase in active zone protein levels in exercised young, adult, and aged NMJs are all localized effects within the exercised muscles and motoneurons innervating exercised muscles (Nishimune et al. 2014).

Interestingly, in human skeletal muscle, resistive exercise training upregulates the expression of laminin β2 at mRNA and protein level (Gordon et al. 2012; Hunter et al. 1989) which, in turn, may play a role in the effect of exercise that increases the level of active zone protein Bassoon in aged NMJs (Nishimune et al. 2012). Laminin β2 is an extracellular matrix protein that is secreted by muscles and is concentrated specifically at the synaptic cleft basal lamina of NMJs (Nishimune et al. 2004; Hunter et al. 1989) which binds specifically P/Q- and N-type voltage-dependent calcium channels (VDCCs) (Nishimune et al. 2004). Several in vivo studies have demonstrated that the interaction between laminin β2 and VDCCs is responsible for organizing the NMJ active zones. The number of active zones, in fact, was decreased when the interaction between the VDCCs and laminin β2 was experimentally perturbed in wild-type mice (Nishimune et al. 2004). Consistent with the above reports, P/Q- and N-type VDCC double-knockout mice exhibited specific defects in the number of active zones and docked synaptic vesicles, which were twice as severe as the defects observed in the P/Q- and N-type VDCC single-knockout mice (Nishimune et al. 2004; Chen et al. 2011). Moreover, humans who carry laminin β2 mutations result in active zone loss and denervation in addition to the development of the “Pierson syndrome,” an autosomal recessive movement disorder associated with microcoria, and nephrotic syndrome (32, 49). These results suggest that laminin β2 binds to synaptic VDCCs to organize the active zones. Recently, studies showed that NMJ active zones are maintained at a constant density as the NMJ matures but are degraded in aged animals (Chen et al. 2012). On the same line of evidence, the expression level of Bassoon decreases in the NMJs of aged mice and rats (Nishimune et al. 2012; Chen et al. 2012). A lack of Bassoon is known to impair synaptic vesicle trafficking to presynaptic membranes in the central nervous system and sensory neurons (Hallermann et al. 2010; Mukherjee et al. 2010; Frank et al. 2010). Furthermore, a lack of Bassoon decreases VDCC Ca²⁺ influx and weakens synaptic transmission, because of the direct interaction between VDCCs and Bassoon that enhances the P/Q-type VDCC function (Nishimune et al. 2012). This modification to VDCCs by Bassoon is similar to the effect of another active zone protein such as RIM1 on VDCCs (Kiyonaka et al. 2007; Uriu et al. 2010). If confirmed, these findings suggest that active zone protein loss may be a part of the molecular mechanism that causes the deterioration of aged NMJs. Active zone deterioration in aged NMJs is ameliorated by muscle exercise. Two months of isometric force training rescued the loss of Bassoon in aged NMJs in the genioglossus muscle of 2-year-old rats (Nishimune et al. 2012). However, exercise training did not alter NMJ size, which suggests that the increase in the Bassoon immunohistochemistry signal in aged NMJs reflects an increase in the protein quantity in each nerve terminal (Nishimune et al. 2012). The mean intensity of the Bassoon immunohistochemistry signal in the NMJs of exercised aged rats is similar to the mean intensity observed in young adult rats. Importantly, the improvement in Bassoon protein level observed in exercised and aged NMJs is consistent with improvements observed using electrophysiology in NMJ function after endurance training in aged mice (Fahim 1997). In summary, exercise-induced upregulation of laminin β2 may play a role in preservation of active zones in aged NMJs, which more likely exerts a positive effect on NMJ synaptic transmission.

Since the beginning of the “human space program,” most of the space research activity has dealt with the effect of μG on bone and skeletal muscle adaptation. For skeletal muscle in particular, most of the studies were made primarily at the physiological and behavioral levels, while only few studies so far investigated molecular and biochemical NMJ adaptation to μG exposure. Nevertheless, these studies however reported that μG during spaceflight induced disturbance in neuromuscular transmission together with a significant decrement in muscle strength (Deschenes et al. 2005).

Data recently available on the histologic and ultrastructural level generally reported that one of the most disruptive NMJ adaptations to μG is axonal disintegration and substantial decrease in synaptic vesicle density and neurotransmitter content, although small quantitative differences were often present between different types of muscle (Baranski and Marciniak 1979; Deschenes et al. 2005). Changes in NMJs of antigravity postural muscles such as soleus (SOL) or adductor longus, as reported, were always more pronounced than other muscles such as extensor digitorum longus (EDL) or gastrocnemius (GAS) (Riley et al. 1990). Interestingly, by using in vivo animal experiments, it was proposed that resistance training in rat SOL is able to remodel structure and size of the NMJ by increasing nerve terminal branching during muscle unloading (Deschenes et al. 2000, 2006). Moreover, neuromuscular adaptations in spaceflown rats were further investigated (Deschenes et al. 2005), which confirmed that these changes are present in particular in postural muscles or antigravity muscles such as the soleus (SOL) and were clearly manifested at the neuromuscular synapse. Thus, the general emerging picture is that under exposure to μG, NMJ experiences disruptive structural adaptations similar to those observed in several models of muscle disuse atrophy on ground such as bed rest inactivity, limb immobilization, denervation, or animal model of hind-limb unloading. Such muscle adaptations might be further accompanied at the level of NMJ by molecular modifications of specific cell signaling pathways and scaffold adapter proteins particularly involved in the synaptic transmission.

So far there are several studies suggesting the importance of Homer protein family in skeletal muscle physiology (Stiber et al. 2005). For instance, specific isoforms of the Homer family have been proposed to directly modulate the intracellular calcium release channel ryanodine receptor 1 (RyR1) activity (Feng et al. 2008). Moreover, binding of Homer modulates the activity of various Ca²⁺ channels, whereas the formation of multimers allows cross-talk between different surface membrane receptors and Ca²⁺ channels in the membrane of intracellular compartments.

Primarily Homer proteins were originally isolated from central nervous system (CNS) neurons as one of the several proteins whose expression is upregulated upon synaptic activity induced by seizure or during induction of long-term potentiation (Shiraishi-Yamaguchi and Furuichi 2007). Homers can bind proline-rich regions present on target proteins through their EVH1 domains and cross-link them through their coiled-coil domains discussed in a recent review (Salanova et al. 2013). The Homer short forms, including Homer1a, the IEG, that directly modify synaptic plasticity (Shiraishi-Yamaguchi and Furuichi 2007) cannot form multimers, but they can still bind to target proteins through their EVH1 domain thereby modifying protein function. Thus, Homer1a disrupts signaling complexes, and therefore, this protein isoform has been proposed to function by acting as a negative regulator of the long forms of Homers (Xiao et al. 1998; Roche et al. 1999).

The EVH1 domain selects a specific consensus binding site within different target proteins that represents a short proline-rich amino acid sequence, PPXXΦ (where X is any amino acid and Φ is any aromatic amino acid), with charged residues on either side of the sequence (Salanova et al. 2013). Database screening analysis showed that there is a long list of potential candidates of Homers interacting proteins, which contained a poly-proline-rich consensus sequence. Some functional proteins were already shown to interact with Homers, e.g., the G-protein-coupled metabotropic glutamate receptor (mGluR) and several Ca²⁺-signaling proteins, including Shank, PLCb, IP3Rs, TRPC channels, RyR, several L-type Ca²⁺ channel isoforms and the nuclear factor of activated T-cells (NFAT) (Huang et al. 2008).

Homers regulate the myogenic program since the transient expression of specific Homer protein isoforms precedes myoblast fusion and thus myotube formation/skeletal muscle differentiation (Stiber et al. 2005). Further evidence supporting this hypothesis comes from findings indicating that Homer 2 mRNA and protein were found to be transiently expressed at high levels in whole embryo at the stage E14.5, particularly in the brain and hind limbs (Stiber et al. 2005). Further biochemical evidence for an effect of Homer 2b isoform on muscle differentiation was ascertained by examining the expression of RyR1 and of the myoglobin promoter, two well-established muscle-specific differentiation markers. Moreover, C₂C₁₂ cells (mouse cell line) expressing Homer 2b augmented basal NFAT transactivation and myoglobin promoter in the order of 2.5-fold greater than of empty vector transfected control cells. Interestingly, co-expression of both activated calcineurin and Homer proteins resulted in a synergistic activation (16-fold) of the myoglobin promoter (Stiber et al. 2005). A proposed mechanism was that Homer might play a role as a molecular switch mediating both IP3R and RyR signaling to promote myotube differentiation program through the NFAT signaling pathway. Thus, Homer proteins potentially represent the first example of a family of scaffolding proteins, which are involved in modulating Ca²⁺-dependent gene expression during muscle differentiation (Stiber et al. 2005).

An additional role of Homer protein family concerning the regulation of excitation-contraction (E-C) coupling was also proposed and suggested by the presence of a Homer binding motif in all RyRs and voltage-dependent L-type Ca²⁺ channels. This hypothesis was further confirmed by specific Ca²⁺ release measurement experiments in permeabilized isolated skeletal muscle fibers as well as by the analysis of the open probability (Po) of the RyRs channels inserted into lipid bilayers, which showed that Homers are able to activate both types of ryanodine receptor Ca²⁺ channels RyR1 and RyR2. Other studies reported that co-expression of Homer1a with RyR2 and Ca_v1,2 enhances, whereas co-expression of Homer1b with RyR2 and Ca_v1,2 reduces, the efficiency of Ca²⁺-induce Ca²⁺ release (CICR) in an in vitro cell model (Huang et al. 2007).