Mechanisms regulating myoblast fusion: A multilevel interplay

Seminars in Cell and Developmental Biology xxx (xxxx) xxx–xxx

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Mechanisms regulating myoblast fusion: A multilevel interplay Lilya Lehka, Maria Jolanta Rędowicz* Laboratory of Molecular Basis of Cell Motility, Nencki Institute of Experimental Biology, Polish Academy of Sciences, 3 Pasteur St., 02-093, Warsaw, Poland

ARTICLE INFO

ABSTRACT

Keywords: Adhesion Cell-cell fusion FKHR MAPK Migration Membrane fusion Muscle pathology Myoblast Myotube NFAT Skeletal muscle SRF Wnt/β-catenin

Myoblast fusion into myotubes is one of the crucial steps of skeletal muscle development (myogenesis). The fusion is preceded by specification of a myogenic lineage (mesodermal progenitors) differentiating into myoblasts and is followed by myofiber-type specification and neuromuscular junction formation. Similarly to other processes of myogenesis, the fusion requires a very precise spatial and temporal regulation occuring both during embryonic development as well as regeneration and repair of the muscle. A plethora of genes and their products is involved in regulation of myoblast fusion and a precise multilevel interplay between them is crucial for myogenic cells to fuse. In this review, we describe both cellular events taking place during myoblast fusion (migration, adhesion, elongation, cell-cell recognition, alignment, and fusion of myoblast membranes enabling formation of myotubes) as well as recent findings on mechanisms regulating this process. Also, we present muscle disorders in humans that have been associated with defects in genes involved in regulation of myoblast fusion.

1. Introduction Skeletal muscle development, also termed as myogenesis, is a complex-multistep process requiring a very precise, space- and timecontrolled regulation that occurs both during embryonic development as well as regeneration and repair of the muscle. It starts from specification of myogenic lineage (mesodermal progenitors) differentiating into myoblasts, which then fuse into multinuclear myotubes. The next step after myotubes formation is their maturation into myofibers that build up a muscle as well as muscle innervation through the newly assembled neuromuscular junction [1]. Each of these processes is tightly regulated by transcription factors with many of them being muscle specific [2]. Noticeably, similar regulatory mechanisms are involved in regeneration and repair of damaged/injured skeletal muscle [3]. The articles of this special issue are focused on newest achievements on understanding of mechanisms of all steps of skeletal muscle differentiation. Here, we concentrate on the mechanisms regulating one of the key steps of mammalian/vertebrate skeletal muscle differentiation, namely myoblast fusion leading to myotube formation. It should be emphasized that cell-cell fusion is not only specific for myogenic cells, though this process occurs only in a limited number of cell types. In mammals, it concerns sperm and oocytes during fertilization, trophoblasts during placenta formation, and macrophages during giant cells ⁎

and osteoclasts formation [4]. Most of the data on mammalian myoblast differentiation, and in particular fusion, comes from in vitro studies on differentiation of murine myoblast cell line, C2C12, though a growing number of data comes from the in vivo studies [5–7]. 2. Myoblast fusion A step preceding myoblast fusion is activation of quiescent myogenic progenitors into proliferative and differentiation (fusion) competent myoblasts; in skeletal muscles the progenitors are termed satellite cells that reside underneath the basal lamina of adult muscle fiber [8–10]. Myogenic specification and differentiation are coordinated by myogenic regulatory factors (MRF) that are basic helixloop-helix (bHLH) transcription factors: Myf5, MyoD (myoblast determination protein), myogenin and MRF4 [11]. They contain a conserved DNA binding domain that binds to the E box DNA motif and dimerize with other HLH containing proteins through an HLH-HLH interaction [8]. Uncommitted progenitor cells are specified into myogenic lineage through combined actions of Myf5, MRF4 and MyoD whereas differentiation into myotubes is controlled by myogenin, MyoD and MRF4 [12]. In mammals, myoblast fusion is a two-phase process (see Fig. 1). In the first phase, myoblast-myoblast fusion occurs to generate initial

Corresponding author. E-mail address: [email protected] (M.J. Rędowicz).

https://doi.org/10.1016/j.semcdb.2020.02.004 Received 7 January 2020; Received in revised form 7 February 2020; Accepted 8 February 2020 1084-9521/ © 2020 Elsevier Ltd. All rights reserved.

Please cite this article as: Lilya Lehka and Maria Jolanta Rędowicz, Seminars in Cell and Developmental Biology, https://doi.org/10.1016/j.semcdb.2020.02.004

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Fig. 1. Regulation of mammalian myoblast fusion. A, Myoblast fusion takes place in two stages: in the first phase myoblast-myoblast fusion occurs to generate the initial multinucleated cells with a limited number of nuclei. In the second phase, additional myoblasts fuse with already formed multinucleated myotubes. B, Formation of a fusion pore. Best characterized molecules and pathways involved in regulation of myoblast fusion are presented on the schemes (see also Table 1). Further explanations in the text.

multinucleated cells then transforming into nascent myotubes. This type of fusion occurs during embryonic development as well as in muscle regeneration and hyperplasia. In the second phase, additional myoblasts fuse with already formed multinucleated myotubes leading to an increased number of myonuclei within the growing myotube. Through the addition of mononuclear cells, the myotube increases its cytoplasmic volume and protein synthesis, thus leading to the myotube size increase. The second phase is most predominant as it takes place not only in embryonic development, regeneration and hyperplasia but also in muscle growth after atrophy as well as in growth and in maintenance of myofibers throughout the life of an individual [13]. The main cellular events taking place during myoblast fusion are migration, adhesion, elongation, cell-cell recognition, alignment, and fusion of myoblast membranes enabling formation of multinuclear nascent myotubes differentiating into mature myotubes (see Fig. 1, [14]). Noticeable, the main cellular events associated with myoblast fusion appear to be identical both in Drosophila and vertebrates/

mammals [15]. Taking into consideration the complexity of muscle tissue, the process of fusion must be tightly regulated by a complex network of signaling pathways to prevent forming abnormal, nonfunctional myofibers. A plethora of genes and their products is involved in regulation of the myoblast fusion and a precise multilevel interplay between them is crucial for myogenic cells to fuse (see Table 1) [12]. 2.1. Myoblast migration Myoblast migration, best described in studies in vitro, enables formation of groups of elongated fusion-competent cells that then align to achieve cell-cell contact and recognition thus promoting myotube formation. Numerous mechanisms are involved in controlling myoblast migration though nearly all of them are not muscle specific. As in other cell types, myoblast migration depends on dynamics of the cytoskeleton, mainly associated with actin and the regulators of its 2

CD164 CXCR4 CXCL12 CCL5 PDGFA FGF2 MRC1 CDH2 CDH15 CAV3 VCAM1 CDON PLEKHO1 PTK2 ADAM12 MMP14 FN1 VTN ITGB1 MAPRE3 FERMT2 MYH9 MYH10 ITGA3 MYMK MYMX ANXA1 ANXA5 MYOF FZD4 PSAP FBLN2 ANK3 TNC NPNT NPHS1 STAB2

CD164 CXCR4 CXCL12 CCL5 (RANTES) PDGF-A b-FGF Mannose receptor N-cadherin

M-cadherin Caveolin-3 VCAM-1 Cdon (Cdo) CKIP-1 FAK

ADAM12 MT1-MMP Fibronectin

Vitronectin β1 integrin EB3 kindlin-2 Non-muscle myosin 2A Non-muscle myosin 2B α3 integrin Myomarker Myomerger (Minion, Myomixer) Annexin-1

Annexin-5

Myoferlin Frizzled-4 Prosaposin Fibulin-2 Ankyrin-3 Tenascin-C Nephronectin Nephrin Stabilin-2

Fusion

Elongation, adhesion and recognition

RAC1 CDC42 DOCK1 DOCK5 MYO6

Rac1 Cdc42 Dock 1 Dock 5 Myosin VI

Migration

Gene name (Homo sapiens)

Protein name

Process

Table 1 Proteins involved in regulation of fusion of mammalian myoblasts.

3 Binding phospholipids in a calcium-sensitive manner Receptor for Wnt proteins Myotrophic factor Binding of fibronectin Membrane-cytoskeleton linker Ligand for integrins Interaction with β1 integrin Regulation of adhesion of mononucleated myoblasts Phosphatidylserine receptor

Binding of phosphatidylserine; myofiber regeneration

Binding of phosphatidylserine

Small GTPase; recruitment of actin and vinculin at contact sites of fusing myoblasts Controlling of rearrangements of the actin cytoskeleton Activation of Rac1 and Cdc42 Activation of Rac1 and Cdc42 Interactions with talin and vinculin; formation of cadherin-dependent cell-cell contacts; inhibition of migration Enhancement of CXCR4-dependent cell motility Chemokine receptor CXCR4 ligand; attractant for CXCR4 muscle precursors Increase of myoblasts migratory activity Enhancement of motility and chemotaxis Enhancement of motility and chemotaxis Regulation of cell migration during myotube growth Cell-cell contact formation; positive regulation of RhoA; negative regulation of Rac1, Cdc42Hs and JNK activities Cell-cell contact formation; activation of Rac1 Caveolae formation; regulation of M-cadherin Facilitation of cell-cell interactions Mediation of cell-cell interactions; interaction with N-cadherin complexes Interaction with phospholipids and BAI1 Regulation of caveolin 3 and β1 integrin expression; modulation of PI3K, mTOR kinase and Erk1/2 kinase signaling Binding of α9β1 integrin Reduction of fibronectin level Extracellular matrix protein; binding to cell surface and various compounds including collagen, fibrin and actin Binding to integrins CD9 cell surface expression, assembly of sarcomeres Microtubule dynamics regulation; promotion of microtubule growth Promotion and stabilization of adhesive structures Interaction with membrane-associated actin; bipolar shape formation and adhesion Interaction with actin filaments; retraction - prevention of myoblasts over-elongation Myoblast alignment and adhesion Regulation of mixing of the cell membrane lipids (hemifusion) Fusion pore formation and expansion

Function

Cytoplasm, plasma membrane, extracellular matrix Cytoplasm, plasma membrane, extracellular matrix Plasma membrane Plasma membrane Extracellular matrix Extracellular matrix Plasma membrane Extracellular matrix Extracellular matrix Plasma membrane Plasma membrane, cytoplasm

Extracellular matrix Plasma membrane Cytoplasm Cytoplasm Cytoplasm Cytoplasm Plasma membrane Plasma membrane Membrane-associated

Plasma membrane Plasma membrane Extracellular matrix

Plasma membrane Plasma membrane Plasma membrane Plasma membrane Plasma membrane Cytoplasm

Plasma membrane Plasma membrane Extracellular matrix Extracellular matrix Extracellular matrix Extracellular matrix Plasma membrane Plasma membrane

Plasma membrane Plasma membrane Cytoplasm Plasma membrane Cytoplasm

Localization

(continued on next page)

[153] [100] [154] [92] [92] [92] [96] [102] [112]

[57]

[7,56]

[147] [148] [149] [150] [35] [35] [151] [49,50] [152]

[144] [145] [146]

[142] [118] [142] [12,30] [31] [143]

[139] [139] [139] [20] [20] [20] [140] [141]

[114] [114] [17] [17] [18,36]

Reference

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organization into more complex actin-rich structures (i.e. filopodia, lamellipodia, podosomes and stress fibers), and contractile properties of the acto-myosin within the stress fibers, anchored to proteins of subplasma membrane adhesion complexes [16]. The main players are Rho family small GTPases (RhoA, Rac1 and Cdc42) responsible for different subcellular actin assembly that target among others Arp2/3 complex involved in filament branching and formins involved in filament bundling [16]. Also, proteins involved in regulation of small GTPase activity such as DOCKs (dedicator of cytokinesis) that are a guanine nucleotide exchange factor (GEF) for Rac1 and Cdc42 were shown to be important for myoblast fusion in mice [17]. Our recent studies on C2C12 myoblasts with a knockdown of unconventional myosin VI showed that this actin-based motor is also involved in myoblast migration as the modified cells were more oval and migrated slower [18]. Myoblast migration is mediated by positive modulators of cell migration such as interleukin 4, CD164 receptor, CCL5 (C-C chemokine ligand 5), PDGF-A (Platelet-derived growth factor subunit A), FGF (Fibroblast growth factor) (see Table 1) [19,20]. Also, sphingosine-1 phosphate and prostacyclin GI2 are involved by playing a role of brakes for migration to enhance cell-cell contact and adhesion [21,22]. The equilibrium between these modulators of cell migration is significantly important for the formation and growth of myotubes, and finally myofibers [3]. It should be emphasized that in vivo myoblasts have to migrate/ penetrate through the basement membrane and connective tissue barriers such as the endomysium and perimysium [23]. And here is the role for matrix metalloproteinases (MMPs), members of a family of zincdependent proteolytic enzymes that are able to degrade extracellular matrix constituents thus facilitating migration and tissue remodeling [24]. MMPs are highly expressed in skeletal muscles and their inhibition results in significant reduction of myoblast migration [25]. So far several MMPs including MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP13 and MMP-14 were shown to stimulate myoblast migration [24–26]. For example, overexpression of MMP9 resulted in improvement of myoblast migration and engraftment [27]. Also, overexpression of MMP-1 or MMP-2 and MMP-13 drastically increased the migration of myoblasts in vitro [26,28]. However, little is known about mechanisms regulating expression of MMPs in myogenic precursors.

Gene and protein annotations were performed with the use of Uniprot database (https://www.uniprot.org/).

[115,116] [129,130] [129,130] [55] [55] [55] [55] Cytoplasm Plasma membrane Plasma membrane Plasma membrane Plasma membrane Cytoplasm (endoplasmic reticulum) Cytoplasm (cytoskeleton) Formation of cell projections Structural component of cell-cell junctions; regulation of Ca2+ homeostasis Structural component of cell-cell junctions; regulation of Ca2+ homeostasis Vesicle trafficking, exocytosis and membrane repair Cell membrane, binding of phosphatidylserine Regulation of the voltage-sensitive calcium channel CACNA1S Actin-associated protein DBN1 PANX1 PANX3 DYSF TRIM72 JSRP1 SYNPO2L Drebrin Pannexin-1 Pannexin-3 Dysferlin Trim72 Jsrp1 Synopo21, CHAP

Localization Function Gene name (Homo sapiens) Protein name Process

Table 1 (continued)

Reference

L. Lehka and M.J. Rędowicz

2.2. Myoblast elongation, adhesion and cell-cell recognition The process of elongation includes interactions between different types of molecules including integrins, matrix remodeling enzymes and a panoply of cytoskeletal proteins (see Table 1). Myoblast elongation is accompanied by formation of filopodia and lamellipodia, cell protrusions which are the actin-rich sites of location of adhesion complexes and are crucial for cell directional movement. It is proposed that these cell extensions also take part in recognition of other myoblasts during fusion as well as response to chemoattractants produced by other cells present in muscle such as for example adipocytes [3]. For fusion, the elongated myoblasts have to adhere to the surface, and transmembrane proteins on each of the cell must properly recognize each other. Various membrane proteins regulating cell-cell contact formation and recognition are involved in this process including N- and M-cadherins, caveolin-3, N- and V-CAMs (neural and vascular cell adhesion molecules of integrin family, respectively), and classical integrins (see Table 1) [29]. N-cadherin is a target of Cdo (termed also as Cdon), a protein serving as multifunctional cell-surface coreceptor with a long extracellular domain containing the multiple IgG-like and FNIII repeats, a single transmembrane domain and a long cytoplasmic domain without similarity to any other known protein [30]. Intracelluar part of Cdo binds to several adhesion molecules and signaling receptors, and extracellular part interacts with N-cadherin complexes of adjacent myoblast to promote assembly and activation of signaling complexes around its 4

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cytoplasmic domain [12]. Several other proteins were found to be involved in enhancing cell membrane proximity and examples of them are casein-kinase 2 interacting protein-1 (CKIP-1) binding phospholipids, and brain specific angiogenesis inhibitor 1(BAI1) that belongs to G-protein coupled receptors (GPCR) [31]. Also, several other proteins cooperate with integrins to control myoblasts fusion. Among them is a nonreceptor protein tyrosine kinase, namely focal adhesion kinase (FAK), modulating several molecules essential for regulation of the actin cytoskeleton dynamics and focal adhesion structure organization [32]. FAK is also a regulator of several anti-apoptotic and cell growth pathways such as phosphatidylinositol-3 kinase (PI3K), mTOR kinase and Erk1/2 kinase signalings [33]. Moreover, MAPK kinase and Wnt pathways are involved in myoblast fusion; their involvement is described in chapters 3.3. and 3.5., respectively. Additionally, several other proteins regulating organization of extracellular matrix such as ADAMs (disintegrin and metalloproteinases), MMPs, VCAM-1, fibronectin and vitronectin cooperate with β1 integrins in regulation of myoblast fusion (see Table 1) [13]. Many other proteins involved in intracellular signaling such as βcatenin (involved not only in cadherin-associated cell-cell contact formation but also in Wnt signaling), end binding 3 protein (EB3, involved in regulation of microtubule organization), kindlin-2 (activator of integrins), myoferlin (involved in membrane fusion, repair and trafficking), creatine kinase 2 and diacylglycerol kinase (involved in cell metabolism) as well as syntrophin (a link between the extracellular matrix and intracellular downstream targets the cytoskeleton by interacting with F-actin) accumulate at sites of contacts between two myogenic cells and play pivotal roles in myoblasts fusion (see Table 1) [34]. Also, myosin motors were shown to be important for myoblast fusion. Non-muscle myosin II isoforms IIA and IIB (NMIIA and NMIIB), expressed in all mammalian tissues and resembling in structure and function muscle myosins, drive changes in myoblasts morphology occurring during their alignment and fusion. While NMIIA is more important for bipolar shape formation and adhesion, NMIIB is involved in retraction thus preventing myoblast over-elongation [35]. Recent studies have also shown that unconventional myosin VI seems to be important for cell-cell contact formation as it not only accumulates at the adhesion sites of myoblasts and myotubes where interacts with talin and vinculin but it is also involved in cadherin-dependent cell-cell contacts formation in many other cell types [18,36]. Moreover, overexpression of not active myosin VI mutant caused formation of abnormal thick myotubes with centrally positioned myonuclei, supporting the hypothesis of importance of this motor for myotube formation [18].

[38]. Examples of non-myoblast fusogenic machineries include SNAREmediated intracellular vesicle fusion [39,40], virus-cell fusion triggered by influenza hemagglutinin (HA) [41–44] and developmental cell fusion of epithelial cells in C. elegans driven by Eff-1 (Epithelial Fusion Failure-1) [45]. Intriguingly, despite numerous studies very little is known about mechanisms of the myoblast membrane fusion. The breakthrough was recent discovery of two essential muscle-specific myoblast fusion proteins, Myomaker and Myomerger (also known as Minion or Myomixer) that together reconstitute fusion in otherwise non-fusing cells suggesting that these proteins catalyze the membrane fusion reaction in myoblasts [46–50]. Genes encoding these proteins (MYMK and MYMX for Myomaker and Myomerger, respectively) are highly expressed in myoblasts and myotubes, and in developing myotome (from embryonic day 10). However, their expression was not detected after postnatal day 21 and in adult myofibers. Interestingly, activation of genes encoding Myomaker and Myomerger takes place in injured muscle [51]. Moreover, disruption of these genes causes embryonic death due to the absence of multinucleated muscle fibers [46,47]. Also, mutations within MYMK in humans cause a congenital myopathy (see chapter 4) [52]. Interestingly, MYOD and myogenin, the key myogenic regulatory factors, can bind to a conserved E-box located proximal to the Myomaker transcription start site and induce its transcription [53]. Additionally, miR-140-3p can inhibit Myomaker expression and myoblast fusion, at least in part, by binding to the 3′ UTR of Myomaker in vitro. These findings confirm the essential role of Myomaker in avian myoblast fusion, and show that MYOD, myogenin and miR-140-3p can regulate Myomaker expression. Myomaker is a 221 amino acid protein with seven transmembrane domains, which exhibits little homology to other characterized fusion proteins [47]. It is required on both fusing cells, and fibroblasts overexpressing Myomaker fuse to myoblasts but do not fuse to each other thus indicating involvement of Myomaker in activation of fusion-competence [48]. Myomerger is an 84 amino acid protein that induces fusion of Myomaker+ fibroblasts and is required in only one of the two fusing cells [48,49]. Myomerger, similarly to Myomaker, harbors little homology to classical fusogens, although there are some similarities with fusion-associated small transmembrane (FAST) proteins expressed by several non-enveloped viruses, which utilize cell-cell fusion to spread the infection [54]. While it is clear that Myomaker and Myomerger are necessary and sufficient for cellular fusion, however, molecular mechanisms by which Myomarker and Myomerger act and cooperate in the myoblast membrane fusion are not fully understood. It has been recently shown that Myomaker and Myomerger independently mediate distinct steps in the fusion pathway, where Myomaker is involved in mixing of membrane lipids (hemifusion) and Myomerger is necessary for fusion pore formation [50]. It was also demonstrated that Myomerger is required on the cell surface where its ectodomains stress membranes and drive fusion completion in a heterologous system independent of Myomaker, and thus a Myomaker-Myomerger physical interaction is not required for function. One of ongoing hypothesis is that both proteins could act through interactions with other proteins [55]. More about this in chapter 3.6. It was also shown that isoforms A1 and A5 of annexin, a membrane Ca2+-binding and phosphatidylserine(PS)-binding protein, facilitate myoblast fusion in culture in a functionally redundant manner [7,56]. In vivo studies revealed that annexin A5 is important in cell fusion required for myofiber regeneration and not in intracellular vesicle fusion needed for repair of myofiber sarcolemma [57]. It should not be forgotten that also lipids such as PS, lysophosphatidylcholine (LPC), prostaglandins (PG), very long fatty acids (VFAT) as well as their position at the plasma membrane and relative content play a crucial role in the membrane fusion [14,58,59]; more about their involvement in myoblast fusion is described in chapter 3.7.

2.3. Membrane fusion Last step of myoblast fusion is coalescence of the cell membranes leading to association of two membranes and volumes they surround. Membrane fusion is fundamental for multiple biological processes including exocytosis, viral infection, and development of syncytial tissues such as striated muscles [37]. A series of distinct membrane events must occur for two cells to fuse and merge their cytoplasmic contents. First, as it was described above, two cells must recognize and adhere to one another, followed by a close membrane adhesion within a distance of about 10 nm. Then, outer-membrane leaflets must fuse, which results in mixing of lipids and the formation of an unstable membrane stalk intermediate (Fig. 1). The formation and expansion of a fusion pore within the hemifused membrane then completes the reaction. The membrane fusion is aided by protein fusion machineries that enable to overcome the extremely high energy barrier to bring membranes in proximity smaller than 10 nm [38]. Interactions between fusogenic protein(s) anchored in each of the membranes, where the proteins undergo a conformational change, lead to remodeling of the membrane phospholipid bilayer and in consequence bring the membranes together 5

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3. Mechanisms regulating myoblast fusion

not expressing IL-4Rα were not able to be recruited by nascent myotubes that secreted IL-4. However, IL-4Rα−/− nascent myotubes fused with wild type myoblasts. During the second phase of fusion that is under the control of NFATC2, the level of IL-4 expression by nascent myotubes increases leading to their fusion with myoblasts through IL4Rα subunit [67]. Mechanisms by which IL-4 controls the process of myogenic cell fusion is not fully understood yet. But there is an evidence that IL-4 could carry out this process by increasing the expression of integrins such as VCAM-1 or VLA-4 (very late activation antigen 4) or by regulating chemotaxis (Fig. 1) [76–78]. Antibodies against VCAM-1 or VLA-4 inhibit myoblast fusion in vitro [79]. The level of VLA-4 was increased during myotubes formation, while VCAM-1 is steadily expressed on the membrane of myoblasts as well as on nascent and mature motubes. VLA-4 - VCAM-1 interaction could play important role in alignment and myoblasts fusion during second phase [4]. Taking into consideration the importance of NFATC2 in skeletal muscle cells, the activators of this pathway could also be involved in myogenesis. The studies have revealed the important role of prostaglandins in muscle functioning [80–82]. Prostaglandins (PGs) are acidic lipids originated from arachidonic acid that are enzymatically produced by mammalian cells in response to mechanical, chemical and immunological stimuli. They are divided into four classes of PG according to the functionalities present in the cyclopentane moiety: PGE, PGA, PGB and PGF. They participate in paracrine signaling. It was shown that PGs induce an increase of intracellular Ca2+ concentration in muscle cells [83]. Following the discovery of PGs, it was shown that prostaglandin F2α (PGF2α) activates calcium dependent transcriptional factor NFATC2 during myogenesis [75]. Further studies devoted to PGF2α-NFATC2 pathway, demonstrated that PGF2α regulates the second phase of myogenic cell fusion. NFATC2 activation is mediated by PGF2α receptor, which level is significantly increased during myogenic cell fusion (Fig. 1). PGF2α receptor is expressed in nascent myotubes, and promotes the fusion of myoblasts with nascent myotubes [83]. The signals for production of PGF2α isoform by myogenic cells have not been revealed yet, but it is assumed that physical activity could be a potent signal leading to PGF2α release [84,85]. One of the group of proteins that are involved in myoblast fusion and are associated with NFAT signaling are ferlins, proteins involved in membrane fusion repair and trafficking, also in myogenic cells where muscle specific isoform, myoferlin, is expressed (Fig. 1) [86]. Myoferlin is associated with cell membrane and binds phospholipids in the Ca2+dependent manner [87]. It was shown that NFATC1 and NFATC3 tightly regulated the myoferlin promoter. Also, mice lacking myoferlin exhibited phenotype similar to NFATC2-null mutants. Myofibrils isolated from these animals were reduced in size and possessed decreased cross-sectional area resulting from fusion defects and delayed muscle repair [88]. Expression of myoferlin is high in myoblasts before initiation of the fusion process. It could be explained in terms of that during myogenic differentiation when the activity of calcineurin is increased, NFATC3 translocates to the nucleus, binds to myoferlin promoter in early myoblast growth stage, preparing them for fusion with myotubes. The role of NFATC1 in myogenesis in vivo is not well established as NFATC1 knockout mice die in utero from cardiac pathology [68].

3.1. Ca2+ involvement and NFAT signaling As in other fusion systems, fusion of myoblast is strongly dependent on the concentration of intracellular Ca2+ ions. It was shown that a decrease in the Ca2+ ions concentration from 1.4 to 0.27 mM resulted in inhibition of rat myoblasts fusion [60,61]. This could be associated with inhibition of acto-myosin contractility and thus migratory functions that are strongly dependent on Ca2+-calmodulin-based regulation [16]. Ca2+ ions can be also implicated in discrete processes which take place during fusion, for example, in the cell-cell and cell-surface contact formation by cadherins. Moreover, the increase in intracellular calcium can activate the calmodulin-dependent serine/threonine phosphatase, calcineurin. Calcineurin-dependent pathways have been implicated in expression of numerous muscle-specific genes [62,63]. This is due to the fact that one of the downstream targets of calcineurin is NFATc, the nuclear factor of activated T cells (Fig. 1) [64]. Members of NFAT family (NFATC1-C4) are calcium sensitive transcriptional factors playing a key role in transcription of genes during immune response by regulation expression of numerous interleukins (IL) such as IL-2, 3, 4, 5 as well as tumor necrosis factor alfa (TNFα) [4,65]. NFATCs are highly expressed not only in T-cells. They are involved in different physiological processes and molecular pathways in different tissues, including skeletal muscles [66–69]. In unstimulated mammalian cells these proteins are located in the cytoplasm in phosphorylated form. As mentioned above, the increase of intracellular Ca2+ ions concentration results in activation of calcineurin that dephosphorylates NFAT, allowing its nuclear translocation and thus evoking induction of transcription of genes controlled by this transcription factor [70]. Of note, there is also NFAT5, which differs from the conventional NFAT proteins NFAT1-4 in its structure, DNA binding, and regulation. In particular, NFAT5 is a constitutively nuclear phosphoprotein regardless of calcineurin activation [71]. It was demonstrated that on distinct stages of myogenesis (myoblasts, nascent or mature myotubes) three isoforms of NFAT (NFATC1, NFATC2, NFATC3) are predominantly expressed. However, each isoform translocates to the nucleus of myogenic cell at the certain phase of muscle development. NFATC3 is expressed in myoblasts and myotubes, NFATC1 and NFATC2 in nascent myotubes, and NFATC2 in myotubes [72,73]. The ability of specific isoforms of NFAT to activate the transcription of genes at certain stages of myogenesis indicate that the individual NFAT isoforms regulate distinct subset of genes during muscle development. Another study demonstrated that soleus muscle isolated from NFATC2 knockout mice was significantly smaller as was shown by a decrease in the myofiber cross-sectional area [4]. The alteration of myotubes growth was also observed in vitro in myotubes lacking NFATC2 as these cells were smaller in size and possessed reduced amount of nuclei in comparison with their normal counterparts. The nuclei number was also decreased in myofibers of NFATC2−/− mice in vivo. These observations point to the fact that initial steps of myoblasts fusion take place normally, but there are aberrations in fusion of myoblasts with nascent myotubes. Thus myoblast (nuclei) addition and further growth of myotubes seem to be controlled by a molecular pathway regulated by NFATC2 [74,75]. Genes regulated by transcriptional factor NFATC2 are the candidates for a new class of regulators of myoblast fusion and muscles growth. It was demonstrated that IL-4 is a downstream target of NFATC2 in muscle cells as antibodies against this cytokine abolished the ability of wild-type conditioned media to induce growth of mutant cells (Fig. 1). Furthermore, IL-4 is not expressed in NFATC2−/− muscle cells compared to wild type cells [75]. To control cell fusion, IL-4 acts through the IL-4Rα receptor on myoblasts but not on myotubes. Myogenic cells lacking IL-4 or its receptor are characterized by the reduction in size and the amount of nuclei. Both myoblasts and nascent myotubes express IL-4Rα subunit on the membrane surface. Myoblasts

3.2. FKHR signaling Another pivotal transcriptional factor regulating myogenic cell fusion is transcription factor FKHR (Forkhead in human rhabdomyosarcoma), also known as FoxO1 (Forkhead box-O1) (Fig. 1) [89]. The main function of FKHR in different studied cell lines is regulation of cell cycle arrest by increasing the expression of cycline-dependent kinase inhibitor p27Kip1 [90]. Activity of FKHR is directly regulated by phosphorylation on Thr24/Ser256/Ser319 residues by AKT-mediated protein kinase B α (PKB α) [91]. It was shown that the level of active FKHR 6

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was increased in nuclei of myoblasts preparing for fusion process, and inhibition of its transcriptional activity resulted in reduction of myoblast fusion. The genes, which protein products were shown to be significantly upregulated under FKHR transcriptional factor, take part in cell fusion (for example frizzled-4 and prosaposin) and extracellular matrix remodeling (for example fibulin-2, ankyrin-3 and tenascin-C) (Fig. 1) [92]. Furthermore, it was demonstrated that promoters of the all abovementioned genes have FKHR binding site, indicating direct regulation of their expression by FKHR [93].

3.5. WNT/β-catenin canonical signaling WNT/β-catenin canonical pathway is essential for controlling embryonic development and adult homeostasis. Wnt proteins interact with Frizzled receptors and low-density lipoprotein receptor-related protein coreceptors (LRP) and stimulate phosphorylation of Disheveled and inactivate GS3Kβ's phosphorylation of β-catenin. This leads to stabilization and nuclear translocation of this cytoskeletal protein. In the myoblast nucleus, β-catenin binds to TCF/LEF1 transcription factors that are involved in activation of myogenic factors such as Myf5 and MyoD [99]. In the absence of Wnt, β-catenin is constantly degraded by the axin complex, consisting of axin, APC (adenomatous polyposis coli protein), CK1 (casein kinase 1) and GSK3 (glycogen synthase kinase 3) [100]. CK1 and GSK3 sequentially phosphorylate the amino terminal region of β-catenin, resulting in β-catenin recognition by β-Trcp (E3 ubiquitin ligase subunit) and subsequent β-catenin ubiquitination leading to proteasomal degradation [100]. This process prevents β-catenin from reaching the nucleus and represses Wnt target genes. [101]. Recently, an important role of WNT/β-catenin signaling for both embryonic muscle development and postnatal myogenesis through regulation of expression of nephrin was demonstrated. Nephrin is a cell surface protein of ephrin family associated with the maintenance of the filtration barrier in the kidney. However, expression of this protein is drastically increased during mouse skeletal muscle development and myoblast fusion. Lack of nephrin resulted in poorly developed muscles and incompletely fused myotubes [102]. Co-cultures of unaffected human fetal myoblasts with nephrin knockout myoblasts or myotubes restored the formation of mature myotubes. Moreover, wild type myoblasts fuse efficiently with nascent nephrin−/− myotubes, indicating that expression of this protein is necessary for mononucleated myoblasts but not for nascent myotubes Following these observations, it was demonstrated that the promoter region of gene encoding nephrine contains a putative WNT/β-catenin response element conserved in all eight species examined (mouse, rat, dog, horse, chimpanzee, orangutan, and human) and expression of this gene was induced after activation of WNT/β-catenin signaling pathway [103]. Several reports describe the alterations in WNT/β-catenin signaling in multiple malformations and syndromes, including muscle disorders in humans [104–106]. It is still unclear how does this signaling pathway regulate myogenesis in a temporal- and spatial-specific manner. One of the mechanisms could be induction in dystrophic muscles of miR-199a, upregulation of which has pro-proliferative effect in numerous normal and transformed tissues [107].

3.3. MAPK signaling MAPK (mitogen-activated protein kinase) pathway plays an essential role in connection of extracellular signals with the changes in transcriptional program. It was shown that the amount of one of the member of this kinase subfamily, namely extracellular signal-regulated kinase 5 (ERK5) significantly increased within minutes during incubation in medium with low serum that it is sustained during terminal myoblast differentiation [94]. Activation of ERK5 affects the E-box dependent transcription. ERK5 increases transcription of p21Cip1 (cyclin-dependent kinase inhibitors) and MLC1A (myosin light chain 1A, a subunit of skeletal muscle myosin). It was also observed that myogenic factors such as MyoD and Mef2C (myocyte-specific enhancer factor 2C) were phosphorylated by ERK5, leading to enhanced transactivation of Mef2C. In C2C12 myoblasts transcribing an antisense RNA for ERK5 caused a block of formation of multinucleated myotubes [94]. It was also shown that ERK1/2 and ERK5 are critical for primary myoblasts fusion. Inhibition of ERK5 pathway had a dramatic effect on this process resulting in formation of mononucleated myoblasts [95] The mechanism, by which ERK5 regulates myoblast fusion is activation of SP1 transcriptional factor (specificity protein 1) (Fig. 1). Other players in this signaling module are genes Klf2 and Klf4 (Kruppel-like factors), possessing in their promoters SP1 binding sites. The amount of Klf2 and Klf4 increases in an ERK5-dependent manner. Overexpression of Klf2 or Klf4 significantly increases the extent of myoblast fusion and opposite, their depletion inhibits fusion. Interestingly, the ERK5/Klf pathway regulates a number of genes involved in fusion, but the increased activity of ERK5 or Klf2 and Klf4 alone cannot accomplish cellcell fusion. They promote this process only in cooperation with myogenic transcription factors, such as MyoD and Mef2, and other proteins essential for fusion such as VCAM-1, cadherin Cdh15, glycoprotein CD164, tertaspamin CD9 and regulator of caveolae, Ehd2 (ATPase EH domain-containing 2) [13]. Another gene, encoding protein nephronectin, also has Klf2 and Klf4 binding sites. Nephronectin is a cellular matrix protein involved in adhesion along with β1 integrin. The amount of nephronectin is significantly increased during the first phase of myoblast fusion [96].

3.6. Myomaker- and Myomerger-based interactions with pathways involved in myoblast fusion

3.4. Serum response factor (SRF) signaling

Due to the fact that Myomaker and Myomerger are small proteins lacking functional domains, the hypothesis has been proposed that both of them could be involved in interaction with other proteins/pathways acting in myoblast differentiation [55]. There are very few data supporting this new notion and the examples are described below. Immunoprecipitation associated with mass spectrometry analysis studies in C2C12 myoblasts identified dysferlin, a member of the aforementioned ferlin family involved in membrane repair, vesicle trafficking and exocytosis, as Myomerger interacting partner [49,108]. Numerous proteins are engaged in cell-contact cites formation such as the ones desribed in chapter 2.2 (see Table 1) but alone they are not sufficient to pull the membranes of fusing cells in close proximity and restricting the membranous lipids. However, they could aid Myomerger and Myomaker in the fusion process [48]. Also, recent studies have shown that Myomaker and Myomerger require actin nucleation, the first step of actin filament formation indispensable for cytoskeleton reorganization that is crucial for myoblast fusion. It was demonstrated that co-expression of Myomaker and

It was shown that serum response factor (SRF) plays a key role in myoblast fusion, in particular in the second phase of the process [97]. Mice with the SRF deletion in skeletal muscle experienced a significant deficiency in skeletal muscle growth and improper maturation [98]. This transcriptional factor stimulates the proliferation and fusion of myogenic cells in a paracrine manner. SRF within myofibers modulates expression of IL-6 and IL-4, which enhance the fusion of myoblasts with nascent myotubes. Nota bene, as it was mentioned above the calcineurin/NFAT pathway also increases expression of IL-4. This is not direct regulation of the aforementioned cytokines by SRF, but SRF enhances the expression of cyclooxygenase-2 (Cox2) gene. Because Cox2derived prostaglandin PGF2a has been demonstrated to control secondary phase of myogenic cell fusion in a NFATC2-dependent manner and IL-4 expression is postulated to be controlled by NFATC2, this transcriptional factor could be the molecule linking Cox2 and IL-4 activation [97]. 7

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Myomerger in fibroblasts resulted in strong cytoskeletal rearrangements leading to formation of actin wall around the cell periphery [49,55]. It is postulated that this effect could be associated with phosphatidylserine signaling, initiation of which activates Rac1 pathway regulating actin polymerization [49]. There are also reports that Myomaker and Myomerger could be regulated by WNT/β-catenin pathway [55]. Recent studies have shown that activation of this pathway resulted in significant up-regulation of the aforementioned dysferlin, believed to be Myomerger interacting partner, as well as three other proteins, synaptopodin-2-like (Synpo2l), junctional sarcoplasmic reticulum protein 1 (Jsrp1), and tripartite motif 72 (Trim72), all of them involved in myoblast fusion when Myomaker and Myomerger work as a complex to ensure fusion pore formation [55].

localizes to cell projections and cell cortex, and in aligned myoblasts and nascent myotubes it concentrates at sites of cell-cell contacts and at the tips of multinucleated cells. RNAi-mediated knockdown of drebrin significantly impaired myotube formation, and expression of myogenin and myosin heavy chains was significantly reduced [115]. Caveolin-3. Caveolin-3 is a scaffolding protein involved in formation of caveolar membranes (caveolae). This protein, involved in signal transduction and vesicular trafficking, interacts directly with G-proteins and can functionally regulate their activity [117]. High expression of caveolin-3 was observed in skeletal and cardiac muscles [118]. It was also shown that C2C12 myoblasts lacking caveolin-3 do not fuse into myotubes [119]. The important role of caveolin-3 for muscle formation and function is further confirmed by association of mutations within its gene with muscle pathology (see chapter 4). Noncoding RNAs. Noncoding RNAs such as microRNAs (miRNA), long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) have been recently found to be critical for muscle physiology, and their aberrant expression is observed in several muscle disorders [120,121]. miRNAs are evolutionary conserved short RNAs transcribed by RNA polymerase II that inhibit posttranscriptional gene expression by inducing mRNA degradation or translational repression [122]. There is over a dozen of miRNAs that were shown to be involved in all steps of myogenesis and skeletal muscle proliferation and only few of them, miR-1, miR-133, miR-135, miR-140-3p, were shown to control myotube formation. It was demonstrated that they miR-133 and miR-135 regulate expression of MAML1 and MEF2C, myogenic transcription factors acting at myoblast fusion [123]. Additionally, miR-133 (one of classical myomiRs) represses the level of SRF, a transcription factor essential for myogenic differentiation [124]. Also, miR-1 (a classical myomiR) by repressing HDAC4 (histone deacetylase 4) synthesis promotes production of follistatin, a protein involved in myoblast fusion that acts as antagonist of myostatin [125,126]. Moreover, as it was mentioned above, miR-140-3p inhibits expression of Myomaker and thus impairs myoblast fusion [53]. lncRNAs share mRNA-like features including polyadenylation and regulate different aspects of gene expression via the mechanisms that are not yet fully recognized [120]. Much less is also known about functions of these molecules in myogenesis in comparison with miRNAs. So far ten lncRNAs were shown to act during myogenesis and skeletal muscle regeneration [120]. One of them, linc-MD1, seems to play a role in myoblast fusion by sponging for miR-133 and miR135 [123]. Interaction of linc-MD1 and miR-133 is enhanced by the RNA binding protein, HuR, and is necessary to maintain myogenic cells in the early differentiation stage. However, elevated level of miR-133 is required for progression into later differentiation stages [127]. The circuitry between linc-MD1, HuR and miR-133 is critical for early differentiation and sponging for miR-133 and miR-135, facilitated by inhibition of HuR activity, is necessary for progression into later differentiation stage [120,127]. Thus these data demonstrate a complex interplay between the miRs and lncRNAs in controlling expression of genes important for myotube formation. Recent studies have also demonstrated that another class of noncoding RNAs, circRNAs, is involved in modulation of gene expression during myogenesis and seems to be associated with muscle-related diseases. These covalently closed continuous loop RNAs are ubiquitously expressed and act as sponges for miRNAs and RNA binding proteins, thus providing an additional mechanism controlling gene expression [121]. They are generated from primary transcripts, predominantly by backsplicing of their exonic, intronic or exon-intron regions [121]. One of several circRNAs acting in muscle is circFUT10, originated from host gene FUT10 (encoding fucosyltransferase 10), that is highly expressed in bovine embryonic skeletal muscle. It has three miR-133a binding sites and thus promotes myoblast differentiation by upregulating the expression of SRF, dependent also on miR-133. CircFUT10 was also shown to promote bovine myoblast apoptosis by affecting expression of Bcl-2 [128].

3.7. Other molecules and mechanisms Lipids. Besides the variety of signaling pathways, transcriptional factors and proteins governing myogenic cell fusion, the properties determined by membrane constitution are also fundamentally important. It is well known that lipids are responsible for membrane fluidity, plasticity and other physicochemical properties of cell membrane. It was observed that the increase of cholesterol to phospholipid ratio reduced the fluidity of membrane because of steroid rings, which immobilize the fatty acids within the membrane. However, unsaturated fatty acids increase the membrane fluidity. The lipid content modifications correlate with the state at which fusion of myoblasts in myotube formation occurs. Very long chain fatty acids (VLCFAs) also have huge impact on membrane plasticity. After incorporation into phospholipid and sphingolipid layers, they promote strong membrane curvature and vesicle fusion [109]. One of the most important role in membrane fusion is assigned to phospholipids, namely to phosphatidylserine (PS) [53,110]. This phospholipid is generally within the inner leaflet of plasma membrane but it translocates to the outer leaflet during apoptosis. This translocation serves as an “eat me” signal for surrounding phagocytes [111]. However, it was shown that PS externalization is also important for myoblast fusion during myogenesis as this phospholipid is transiently exposed at cell-cell contacts [110]. Stabilin-2 (Stab2), the main receptor for PS, is highly expressed in different murine muscles. Stab2 acts as a membrane protein for myoblasts fusion during muscle development. Myoblasts lacking stabilin-2 exhibited lowered phosphatidylserine-dependent fusion. The expression of Stab2 in myoblasts is regulated by calcineurin/NFAT signaling pathway. Following this observation, it was shown, that NFATC1 regulates the transcription of Stab2 gene by direct interaction with its promoter [112]. The recognition of PS in the outer leaflet of plasma membrane by phosphatidylserine receptors induces intracellular signaling resulting in the actin cytoskeleton rearrangement. Actin cytoskeleton dynamics. Besides PS-dependent mechanisms, the cytoskeleton dynamics is regulated by small GTPases of Rho family [113]. These proteins play crucial role during myoblasts fusion by regulation of activity of several kinases and transcriptional factors such as for example ROCK, MAPK, JNK and SRF. Deletion of genes encoding small GTPases Rac1 and Cdc42 resulted in significant reduction of the fusion index and number of nuclei in developing myofibers. The level of proteins regulating actin filament formation such as Vasp (vasodilatorstimulated phosphoprotein) and adhesive structure formation such as vinculin was also reduced in contact sites of myoblasts lacking these GTPases [114]. Drebrin. One of the proteins that has been recently associated with myoblast migration and adhesion is drebrin, a protein involved in the cytoskeleton organization, encoded by DBN1 gene [115]. Its expression is very low in proliferating myoblasts and was induced after shifting into differentiating conditions which is associated with binding of MyoD to Dbn1 locus [107]. In non-proliferating myoblasts drebrin 8

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Pannexins. Pannexins (Panxs) oligomerize into hemichannels that serve as mediators of ATP release by several cell types thus taking a part in cell communication with the extracellular environment as ATP affects Ca2+-wave propagation through nucleotide P2X and P2Y receptors. In skeletal muscle and myogenic cells, Panx1 and Panx3 were found to be expressed and involved in myoblast differentiation and fusion [129]. The amount of Panx1 increases drastically during myoblast differentiation, and inhibition of Panx1 channels significantly reduced differentiation and fusion of human primary skeletal muscle myoblasts [130]. The level of one of Pnx3 immunoreactive forms (∼43 kDa) is very low in myotubes and is higher in skeletal muscle, but its overexpression also promoted differentiation and fusion of the aforementioned myoblasts. In contrast to these observations, the other form (∼70 kDa) is expressed in high levels in undifferentiated myoblasts and is downregulated during differentiation [130]. It is speculated that pannexins may be involved in developing rhabdomyosarcomas, in which processes associated with proliferation and differentiation are deregulated [129]. Exosomes. Exosomes are small vesicles derived from multivesicular bodies and are released from cells during exocytosis and contain cargo composed of proteins, miRNAs and lipids involved in cell-cell and celltissue communication. Exosomes secreted by C2C12 myoblasts and myotubes show enrichment in numerous proteins including aforementioned annexins and Ehds, and were shown to control not only cell myoblast proliferation but also fusion [131–133]. Thus these membranous particles are implicated in a cross-talk between mature muscle/ myotube and myoblasts.

age, apnea and feeding problems. HACD1 plays a key role in synthesis of very long chain fatty acids (VLCFAs) and is mainly expressed in the skeletal and cardiac muscles where its expression is regulated by transcriptional factor SRF, which is responsible for the secondary myoblast fusion [136,137]. Lack of this enzyme leads to changes in the plasma membrane fluidity and degree of saturation thus having strong influence on myoblast fusion. Caveolin-3 associated. Mutations within gene encoding caveolin-3, a protein involved in myoblast fusion, are responsible for different forms of muscle diseases, including limb-girdle muscular dystrophy type 1C (LGMD-1C), hyperCKemia (HCK), distal myopathy (DM), and rippling muscle disease (RMD) [138]. The best characterized is LGMD-1C that is caused by autosomal dominant mutations in caveolin-3 gene. The main clinical features of the patients with caveolin-3 mutations were calf hypertrophy and mild-to-moderate proximal muscle weakness [138]. This could be due to alterations of myoblast fusion due to impairment of activity of mutated caveolin-3. 5. Conclusions Recent studies significantly contributed to broaden the knowledge on molecular mechanisms governing myoblast fusion, and myogenesis in general. The examples are identification of Myomaker and Myomixer, involvement of non-coding RNAs and exosomes, just to name achievements that in our opinion seem to be most spectacular. Also, a progress in technology enabled to perform studies on proteomeand genome-wide scale with the use of several transgenic animal models such as for example Drosophila, zebrafish and rodents as well as cell lines derived from patients with myopathies. Also, improvements of methods enabling life imaging of the fusion process both in vitro and in vivo significantly contributed to understanding how the fusion progresses. Despite the evident advancement in studying the regulatory mechanisms of myogenic cell fusion, this area still remains poorly investigated and further studies are needed, especially with the use of mammalian systems. In our opinion, they should focus on identification of extracellular stimuli that activate genes or leading to the activation of specific pathways involved in regulation of myoblast fusion in health and disease. Also, very little is known whether and how the processes directly following the fusion and myotube formation such as the nucleus positioning, myofiber type specification and neuromuscular junction formation are interrelated with mechanisms governing fusion per se. In-depth understanding of myoblast fusion will not only broaden the knowledge on this crucial step of myogenesis but could also provide the tools for development of novel therapeutic and preventive approaches for muscle developmental defects and for progression of the newly emerging field of tissue-engineering aimed at muscle regeneration and repair.

4. Human myopathies connected with perturbation of myogenic cells fusion Human muscular dystrophies are the group of disorders connected with progressive muscle weakness and wasting that affect axial, limb, facial or other muscles, including cardiac, respiratory, etc. [134]. A plethora of genes encoding transcriptional factors, proteins constituting myofiber and signaling pathways are involved in myogenesis thus a mutation in any of them could be a cause of muscle pathology. Several muscle disorders in humans have been directly associated with defects in specific genes, which are involved in regulation of myoblast fusion. Myomaker associated. One of such disorders is Carey-Fineman-Ziter syndrome (CFZS) that is caused by mutations within a gene encoding Myomaker (MYMK), a protein that is essential for myoblast fusion and formation of multinucleated myotubes in verterbrates (see chapter 2.3.). So far five autosomal recessive mutations were identified that result in the reduced expression of MYMK, and thus patients with CFZS have partially functioning protein. CFZS is a congenital myopathy characterized by marked bilateral facial weakness, generalized muscle hypoplasia, scoliosis and inability to fully abduct both eyes [52]. Muscle biopsies from these patients revealed a marked hypertrophy of both type I and II fibers, and the muscle fiber diameter was twice increased compared to the age-matched controls. A few internalized nuclei and irregularities in the intermyofibrillar network were also shown [135]. Experiments devoted to the rescuing of the mutated MYMK gene in zebrafish seem promising for future therapies, which will be aimed at restoration of the function of this gene in humans [52]. Lipid metabolism associated. Taking into consideration the importance of lipids in regulation of myogenic cell fusion, the disturbances in maintaining of a lipid balance could affect the muscle homeostasis and plasticity. In humans and dogs, the deficiency of 3hydroxyacyl-CoA dehydratase 1 (HACD1) leads to congenital myopathy caused by a mutation in a gene encoding this enzyme. This deficiency, resulting from a nonsense-mediated decay of the HACD1 mRNA to ∼30% of control levels in patient muscles, completely abrogates the enzymatic activity of the protein [136]. The disease is characterized by early postnatal heterogeneity in myofiber size, reduced diameter of skeletal myofibers, general weakness with gradual improvement with

Declaration of Competing Interest There are no conflict of interest. Acknowledgments The authors are grateful to Prof. Malgorzata Daczewska, the Guest Editor of this Special Issue for the invitation to contribute. This work was supported by the grant no. 2017/27/B/NZ3/01984 from National Science Centre, Poland and statutory funds for the Nencki Institute from the Ministry of Science and Higher Education, Poland. References [1] B.C. Syverud, K.W. Vandusen, L.M. Larkin, Growth factors for skeletal muscle tissue engineering, Cells Tissues Organs (Print) 202 (3–4) (2016) 169–179,

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