Overview of skeletal muscle regeneration

In adulthood, muscle regeneration occurs after injuries and resembles in many aspect to muscle development during embryogenesis. Muscle stem cells, called satellite cells, located between the basal lamina and the sarcolemma of muscle fibers are responsible for the capacity of muscle to regenerate (Mauro 1961). They are characterized by specific markers such as Pax7 that are, however, dispensable for proper adult muscle regeneration as muscle from inducible Pax7 KO mice are still able to regenerate (Lepper, Conway et al. 2009). Upon injury, satellite cells become activated, as they received multiple activation stimuli such as nitric oxide, sphingosine-1-phosphate (Nagata, Partridge et al. 2006, Tatsumi, Liu et al. 2006), fibroblast growth factor (FGF) and transforming growth factor-β (TGF-β), insulin-like growth factor (IGF), hepatocyte growth factor (HGF), and interleukin-6 (IL-6) whose synthesis is enhanced after muscle damage (Grefte, Kuijpers-Jagtman et al. 2007). The balance between

satellite cell proliferation and commitment into differentiation is tightly controlled by Notch and Wnt signaling pathways, respectively. This ensures the maintenance of a pool of satellite cells together with proper muscle regeneration (Conboy and Rando 2002, Brack, Conboy et al.

2008). Satellite cells committed into the myogenic differentiation process but still in proliferation are called myoblasts or myogenic precursor cells (MPC). When cell cycle is arrested, they start to fuse and form multinucleated myotubes with central nuclei. Further maturation of these fibers is characterized by size augmentation and nuclei migration at the edge of the fiber. A gradual increase in expression of muscle specific transcription factors belonging to MEF2 (Myocyte Enhancer binding factor 2) and MRFs (Muscle Regulatory factors) families controls each step of the differentiation process (Edmondson, Lyons et al. 1994, Cornelison and Wold 1997, Cornelison, Olwin et al. 2000). Sequential activation of the different MRFs beginning by MyoD and/or Myf5 expression, followed by Myogenin and finally MRF4 together with the different MEF2 isoforms, drive the expression of muscle specific proteins such as MyHC (Myosin heavy Chain) and other proteins of the contractile apparatus (figure III.14) (Sartore, Gorza et al. 1982, Ciciliot and Schiaffino 2010).

Figure III.14: Scheme of myogenesis and markers typical of each stage.

From Zammit P.S., et al. (2006)

Satellite cells are quiescent in normal adult muscle and can be activated by, for example, muscle damage. Once activated, satellite cells divide to produce satellite cell-derived myoblasts that further proliferate, before committing to differentiation and fusing to form myotubes, which then mature into myofibers (for clarity, satellite cell self-renewal is not included). Pax7 and Myf5 are expressed in quiescent satellite cells. Satellite cell activation is marked by the rapid onset of MyoD and MEF2A/D expression, whereas myogenin and MEF2C later mark the commitment to differentiation. The temporal expression pattern of MyHC is typical of many structural muscle genes, which mark sarcomeric assembly in the later stages of differentiation.

4.2.2. From myotubes to contracting muscle fibers

While the first steps of myogenic differentiation can easily be recapitulated in vitro, late events leading to a mature muscle fiber are much more complicated to mimic in a petri dish.

Indeed, the maturation phase is controlled by environmental stimuli such as electrical stimulation, growth factors as well as mechanical load that are still ill-defined and/or difficult to implement in culture. Knowledge of the final step of adult muscle regeneration is hence still poorly investigated and the process that I will described concerning ECC apparatus development in muscle fibers have been mainly decipher from in vivo experiments with animal models during embryogenesis. As previously mentioned, ECC is ensured by 3 muscle components: the sarcomeres, the SR and the triads. Formation of these structures begin independently during muscle development before their assembly throughout the late maturation phases (figure III.15).

Figure III.15: Organization of SR domain in adult skeletal muscle in line with the myofibrils.

Adapted from Rossi D., et al. (2008)

(A) Cartoon depicting SR domains in correspondence to the sarcomere organization. T-tubules (black arrow heads) are organized in a double row pattern, each of them placed at the A-I band interface. J-SR juxtaposed to the t-tubule show the same profile as revealed by RyR1 staining of adult skeletal muscle fibers in (B), middle panel. L-SR (red arrows) correspond to Z-line and M band. Note that SR proteins density is more important at the Z-line than the M-band as highlighted by SERCA labelling in (B), upper panel. RyR1, Ryanodine Receptor 1; SERCA, sarco/endoplasmic reticulum Ca²⁺-ATPase; SR, sarco/endoplasmic reticulum; j-SR and l-SR, junctional and longitudinal SR. Scale bar 5μm.

Even though not completely understood, the main steps of myofibrillogenesis have been unraveled thanks to imaging of fluorescent tagged sarcomeric proteins with FRAP (Fluorescence Recovery After Photobleaching) and FRET technics (Sanger, Wang et al. 2010).

Assembly of myofibrils begins at the edges of muscle cells with cortical actin that nucleates from z-bodies, composed of α-actinin, which prefigure the Z-line. Then N-terminal tails of titin are anchored to the z-bodies and allow the recruitment of myosin units to form the thick filaments. As mature sarcomeres form, myosin-binding proteins such as troponin C and myomesin are incorporated. Myomesin is recruited via the C-terminal domain of fully synthesized titin that spans half of the sarcomere length, to form the M-bands (Myhre and Pilgrim 2014, Gautel and Djinovic-Carugo 2016) (figure III.16).

Figure III.16: The molecular model of myofibrillogenesis.

From Myhre J. L., et al. (2014)

The molecular model, demonstrating the stages of myofibrillogenesis. (A) Cortical actin organizes into stress-fiber-like structures around nucleating α-actinin at attachment sites. N-terminal titin associates with α-actinin in these structures. (B) N-terminal titin stabilizes the Z-disk. As titin is translated, the semi-rigid rod region recruits myosin hexamers, establishing the thick filament core. (C) Fully-formed titin recruits myomesin to the C-terminus, thus establishing the M-line. The length of titin establishes the precise Z-M-Z register in the new myofibrils.

Concerning SR and t-tubule structures, EM studies of mouse embryogenesis allowed important findings about their formation. SR develops from tubulation of the ER whereas t-tubules are progressive invagination of the PM within the entire myofiber. At E14, longitudinal SR expands around myofibrils in a reticular network and is attached to sarcomeres at the level of Z disk and M band (figure III.15). From E15 to E19 t-tubules develop progressively with a longitudinal orientation but are connected to PM via short transverse elements. Even though t-tubule formation is not completely understood, evidences points toward caveolae as starting point in their biogenesis (Carozzi, Ikonen et al. 2000, Galbiati, Engelman et al. 2001).

Caveolae are cholesterol-sphingolipid rich region of the PM showing vesicular aspect. Their shaping is controlled by the caveolin 3 (CAV3) isoform that is mainly expressed in striated muscle (Tang, Scherer et al. 1996). Additionally to CAV3, Amphiphysin 2 (BIN1) a protein able to induce membrane bending and curvature (Razzaq, Robinson et al. 2001, Lee, Marcucci et al. 2002, Toussaint, Cowling et al. 2011), dysferlin (DYSF) implicated in membrane repair (Klinge, Harris et al. 2010) and myotubularin (MTM1) participate also in t-tubule biogenesis (Al-Qusairi, Weiss et al. 2009). In parallel with t-tubules development, an increasing number of connections via dyads and triads take place with the junctional SR (j-SR). J-SR and t-tubule containing RyR and DHPR respectively, first couple at the periphery of the myofiber before connections occur at the A-I band interface with a longitudinal orientation by E17 (Franzini-Armstrong 1991, Takekura, Flucher et al. 2001). Mitsugumin 29 (MG29) and junctophilin 1 (JPH1) that are both transmembrane SR proteins, play a role in triads assembly by ensuring docking of the SR to the t-tubules (Komazaki, Nishi et al. 1999, Nishi, Komazaki et al. 1999, Takeshima, Komazaki et al. 2000, Ito, Komazaki et al. 2001, Komazaki, Nishi et al. 2001, Komazaki, Ito et al. 2002). Eventually, typical t-tubules transverse orientation highlighted by RyR and DHPR double row pattern in immunostaining will occur only 3 weeks after birth in mice (figure III.15).