**4. Inducing muscle hypertrophy as therapeutic strategy for MDs**

In adult muscles, satellite cells are quiescence and blocked in the G0 phase of the cell cycle. After activation, these satellite cells move outside of the basal lamina and express Pax7 and MyoD. These cells are now known as myoblasts. They extensively divide and fuse to differ‐ entiate and form multinucleated myofibres. During this late differentiation process, myoblasts down-regulate Pax7 and express myogenin. MyoD and Myf5 factors are involved in the early stages of transitioning from undifferentiated myogenic precursors to myoblasts [16]. On the contrary, myogenin and MRF4 regulate the transition from myoblast to mature fibres.

In adulthood, a skeletal muscle can enlarge or reduce its own mass through a complex interplay in which several molecules are involved. During skeletal muscle hypertrophy, the myofibrils increase in number and size to increase muscular strength (Figure 3). While sarcoplasmic hypertrophy is a characteristic of body-builders' muscles, myofibrillar hypertrophy is typically found in weight lifters. In contrast, muscle atrophy, also known as muscle wasting, is the result of muscle protein loss with a reduction in fibres. At a molecular level, signals control both muscle growth and atrophy. These are finely interconnected and the biochemical pathways can be altered by increasing or decreasing specific growth factors [17]. Insulin Growth Factor-1 (IGF-1) is the most reliable muscle growth-promoting factor. IGF-1 is largely produced in the liver. However, skeletal muscle also contributes to the production of two distinct IGF-1 isoforms. Different isoforms of IGF-1 exist due to different RNA spliced variants. Human skeletal muscle has been found to express at least two isoforms [18]. These are IGF-1Ea, which is the liver type or systemic form and IGF-1Ec, also called Mecano Growth Factor (MGF). MGF is an autocrine/paracrine form that is particularly interesting as it is expressed in response to mechanical stimuli and cellular damage.

Increased muscle loading leads to augmented expression of the IGF-1 encoding gene, both in humans and animal models. Several authors have indicated that IGF1Ea induces proliferation and differentiation of satellite cells and muscle hypertrophy [19, 20]. Transgenic mice overex‐ pressing IGF-1Ea display, indeed, skeletal muscle hypertrophy associated with increased muscle strength [21].

IGF-1Ea and other isoforms act via a tyrosin-kinase receptor IGFR-1, enabling AKT1 to be activated by the generation of phosphatidylinositol-3,4,5-triphosphates (PIP3). PIP3 Kinase (PI3Kinase) and the phosphatases PTEN and SHIP2 regulate the formation of PIP3, which recruits AKT1 on the plasma membrane. They can activate the mammalian target of rapamycin (mTOR) or FK506-binding protein 12-rapamycin-associated protein 1 AKT1 (PKBa). Further‐ more, mTOR generates two complexes: the Rapamycin-sensitive Ternary complex mTORC1 and Rapamycin-insensitive mTORC2. These complexes control pathways that determine the mass/size (mTORC1) and the shape (mTORC2) of the cells. The general activation of mTOR results in the phosphorylation of several downstream targets in a signalling cascade. In this view, AKT1 is responsible for modulating the muscle growth and protein up-regulation signals in skeletal muscle tissue.

Met-Activating Genetically-Improved Chimeric Factor 1 (Magic-F1) is a recombinant protein that also triggers AKT pathway [22]. Magic-F1 is constructed as a bivalent ligand from HGF,

**4. Inducing muscle hypertrophy as therapeutic strategy for MDs**

mechanical stimuli and cellular damage.

muscle strength [21].

398 Muscle Cell and Tissue

in skeletal muscle tissue.

In adult muscles, satellite cells are quiescence and blocked in the G0 phase of the cell cycle. After activation, these satellite cells move outside of the basal lamina and express Pax7 and MyoD. These cells are now known as myoblasts. They extensively divide and fuse to differ‐ entiate and form multinucleated myofibres. During this late differentiation process, myoblasts down-regulate Pax7 and express myogenin. MyoD and Myf5 factors are involved in the early stages of transitioning from undifferentiated myogenic precursors to myoblasts [16]. On the contrary, myogenin and MRF4 regulate the transition from myoblast to mature fibres.

In adulthood, a skeletal muscle can enlarge or reduce its own mass through a complex interplay in which several molecules are involved. During skeletal muscle hypertrophy, the myofibrils increase in number and size to increase muscular strength (Figure 3). While sarcoplasmic hypertrophy is a characteristic of body-builders' muscles, myofibrillar hypertrophy is typically found in weight lifters. In contrast, muscle atrophy, also known as muscle wasting, is the result of muscle protein loss with a reduction in fibres. At a molecular level, signals control both muscle growth and atrophy. These are finely interconnected and the biochemical pathways can be altered by increasing or decreasing specific growth factors [17]. Insulin Growth Factor-1 (IGF-1) is the most reliable muscle growth-promoting factor. IGF-1 is largely produced in the liver. However, skeletal muscle also contributes to the production of two distinct IGF-1 isoforms. Different isoforms of IGF-1 exist due to different RNA spliced variants. Human skeletal muscle has been found to express at least two isoforms [18]. These are IGF-1Ea, which is the liver type or systemic form and IGF-1Ec, also called Mecano Growth Factor (MGF). MGF is an autocrine/paracrine form that is particularly interesting as it is expressed in response to

Increased muscle loading leads to augmented expression of the IGF-1 encoding gene, both in humans and animal models. Several authors have indicated that IGF1Ea induces proliferation and differentiation of satellite cells and muscle hypertrophy [19, 20]. Transgenic mice overex‐ pressing IGF-1Ea display, indeed, skeletal muscle hypertrophy associated with increased

IGF-1Ea and other isoforms act via a tyrosin-kinase receptor IGFR-1, enabling AKT1 to be activated by the generation of phosphatidylinositol-3,4,5-triphosphates (PIP3). PIP3 Kinase (PI3Kinase) and the phosphatases PTEN and SHIP2 regulate the formation of PIP3, which recruits AKT1 on the plasma membrane. They can activate the mammalian target of rapamycin (mTOR) or FK506-binding protein 12-rapamycin-associated protein 1 AKT1 (PKBa). Further‐ more, mTOR generates two complexes: the Rapamycin-sensitive Ternary complex mTORC1 and Rapamycin-insensitive mTORC2. These complexes control pathways that determine the mass/size (mTORC1) and the shape (mTORC2) of the cells. The general activation of mTOR results in the phosphorylation of several downstream targets in a signalling cascade. In this view, AKT1 is responsible for modulating the muscle growth and protein up-regulation signals

Met-Activating Genetically-Improved Chimeric Factor 1 (Magic-F1) is a recombinant protein that also triggers AKT pathway [22]. Magic-F1 is constructed as a bivalent ligand from HGF,

**Figure 3.** Muscle remodelling is a complex process and is among many key factors that modulate both satellite cells activation and protein synthesis. As a final result, fibre size and nuclear content can be increased (hypertrophy, right panel). In the cartoon, molecules can induce muscle growth and the key players in the hypertrophy-signalling path‐ way are indicated.

containing the signal peptide, the N-domain and the first two kringle-domains K1 and K2 of HGF. However, the kringles repeat in tandem and are joined by a linker. Magic-F1 binds c-Met and the HGF receptor activates Akt but not the Erk signalling pathway. Therefore, this recombinant molecule, which enhances the myogenic differentiation process, is a safe mole‐ cule. It does not have the potential risk of stimulating uncontrolled proliferation observed in several growth factors including IGF1. Previous studies in transgenic mice expressing Magi-F1 under muscle specific promoter showed that the recombinant protein cooperates with Pax3 signal pathway in early embryogenesis. This generates a more active skeletal muscle progen‐ itors in early embryogenesis [23]. This results in a constitutive muscular hypertrophy in the adulthood of transgenic mice, since Magic-F1can down-regulate myostatin, a potent muscle mass regulator. Furthermore, it can directly activate MyoD, Myf-5 and several anti-apoptotic pathways.

The therapeutic potential of Magic-F1 was evaluated on α-sarcoglycan null mice (Scga-/-), an established model of limb-girdle muscular dystrophy type 2D. The Scga-/-/Magic-F1 trans‐ genic mice showed a stronger muscle phenotype than their Scga-/- counterparts. Furthermore, the physiological benefit of muscular hypertrophy partially recovered the dystrophic pheno‐ type [22].

Mutations occurring in the myostatin gene, also named growth and differentiation factor 8 (GDF8), are responsible for a hypertrophic phenotype. There is a great increase in muscle mass in a breed of beef cattle known as Belgium Blue [24]. Mdx mice that do not express myostatin are stronger and more muscular than their mdx counterparts [25]. Fibrosis and fatty remod‐ elling are less evident in the diaphragm of those transgenic mice, suggesting improved muscle regeneration. In 2004, the first mutation of the myostatin gene in humans was reported and correlated with an enlargement of the skeletal muscle apparatus [26].

In summary, inducing muscular hypertrophy is relevant in clinical applications as a potential treatment of muscle diseases, including muscular dystrophy and cachexia, which cause wasting in muscle mass and force. Insulin-like Growth Factor-1 (IGF-1), MAGIC-F1 and myostatin regulate the key steps during muscle regeneration (Figure 3). In animal models for Duchenne muscular dystrophy [22, 25, 27], these molecules have demonstrated a therapeutic value, without redressing the primary cause of the lesion and, in principle, could be adopted in patients suffering from muscular dystrophies. The delivery strategies of these molecules and potential side effects require more investigation. So far, their translational potential has been hindered in clinical trials.
