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Myostatin inhibitor proteins increase muscle mass and strength in wild-type C57BL/6 mice. ( a ) Gross hindlimb muscle mass is increased in all myostatin-inhibitor-protein treated mice at 725 days of age compared with 

Myostatin inhibitor proteins increase muscle mass and strength in wild-type C57BL/6 mice. ( a ) Gross hindlimb muscle mass is increased in all myostatin-inhibitor-protein treated mice at 725 days of age compared with 

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Increasing the size and strength of muscles represents a promising therapeutic strategy for musculoskeletal disorders, and interest has focused on myostatin, a negative regulator of muscle growth. Various myostatin inhibitor approaches have been identified and tested in models of muscle disease with varying efficacies, depending on the age at which...

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... new approaches for postnatal muscle enhancement and expanded the potential for gene therapy to be considered as a method to inhibit myostatin activity. Follistatin (FS) has been shown to bind to some TGF- ␤ family members and can function as a potent myostatin antagonist. Overexpression of follistatin by transgenic approaches in muscle has been shown to increase muscle growth in vivo (13), and a lack of follistatin results in reduced muscle mass at birth (14). Recent data has also shown that follistatin is capable of controlling muscle mass through pathways independent of the myostatin signaling cascade. In these studies, myostatin knockout mice were crossed to mice carrying a follistatin transgene. The resulting mice had a quadrupling of muscle mass compared with the doubling of muscle mass that is observed from lack of myostatin alone, confirming a role for follistatin in the regulation of muscle mass beyond solely myostatin inhibition (15). In addition to follistatin, two other proteins have been identified that are involved in the regulation of the myostatin. Follistatin-related gene (FLRG) is highly similar to follistatin and has been shown to inhibit activin and multiple bone mor- phogenic proteins in vitro (16, 17). Growth and differentiation factor-associated serum protein-1 (GASP-1) is a protein that has been discovered to contain multiple domains associated with protease-inhibitor proteins and a domain homologous to the 10-cysteine repeat found in follistatin. GASP-1 was shown to bind directly to the mature myostatin and myostatin propeptide and inhibits myostatin’s activity (18). Although recombinant protein injections or myostatin blocking antibodies are feasible strategies, gene therapy to express these myostatin inhibitor genes may prove a more efficacious therapeutic route for numerous reasons, including the lack of potential immune response to antibody treatment and the requirement for multiple injections. Here, we report that a one-time postnatal intramuscular injection of adeno-associated virus (AAV) encoding myostatin- inhibitor-proteins resulted in long-term improvement of muscle size and strength in wild-type animals. Delivery of a myostatin- inhibitor-protein in dystrophic mdx animals reversed muscle pathology and improved strength, even when administered in 6.5-month-old animals. Specifically, we show here that follista- tin-344 resulted in the greatest effects on muscle size and function and was well tolerated with no untoward effects on cardiac pathology or reproductive capacity in either male or female treated animals. AAV-mediated gene delivery to muscle provides a system to generate high levels of protein in the target tissue or by a secreted product carried to remote sites through the circulation (19). We cloned the known secreted myostatin-inhibiting genes, including growth and differentiation factor-associated serum protein-1 (GASP-1) (18), follistatin-related gene (FLRG) (17), and follista- tin-344 (FS) (13) into AAV serotype 1, which have demonstrated high muscle transduction capabilities. There are two isoforms of follistatin generated by alternative splicing. The FS-344 variant undergoes peptide cleavage to generate the FS-315 isoform and the other FS-317 variant produces the FS-288 isoform after peptide cleavage. We used the human FS-344 variant, which exclusively generates the serum circulating FS-315 isoform of FS and includes a C-terminal acidic region (20). We chose FS-344 (FS), because the other FS-317 isoform, lacking the C terminus, shows preferential localization to the ovarian follicular fluid and high tissue binding affinity through heparin sulfate proteoglycans, which may affect reproductive capacity and bind to other off-target sites (21). FS-288 represents the membrane-bound form of follistatin (22), is a potent suppressor of pituitary follicle stimulating hormone (23), is found in the follicular fluid of the ovary and in the testes, and demonstrates a high affinity for the granulosa cells of the ovary. We sought to determine the efficacy of these proteins to increase muscle mass in normal and dystrophic mice. We administered 1 ϫ 10 11 AAV1 viral particles per animal encoding FS, FLRG, GASP-1, or GFP bilaterally into the quadriceps and tibialis anterior muscles of 4-week-old wild-type C57BL/6 mice. All animals treated with the myostatin inhibitors demonstrated an increase in body mass with an observable gross enhancement of muscles when analyzed at 725-days of age compared with GFP-treated controls (Fig. 1 a and b ). Evaluation of individual muscle weights showed an increase in muscle mass for all myostatin inhibitor-treated animals, with the greatest increase in FS-treated animals. The increased muscle mass was found in the injected hindlimb muscles and remote muscles to the injection site, such as the triceps. Thus, these inhibitors were secreted into the circulation from the site of muscle injection, enhancing skeletal muscle mass at remote sites (Fig. 1 c ). The enlarged muscle mass was accompanied by functional improvement demonstrated by an increase in hindlimb grip strength (Fig. 1 d ). There was no effect on heart mass or histological appearance of cardiomyocytes, indicating that myostatin inhibition was selec- tive to skeletal muscle tissue (data not shown). There has been concern that FS adversely effects gonadal function. We found no change in reproductive capacity in mice treated with our AAV1 carrying the FS344 transgene (AAV1-FS, Table 1) Furthermore, we found no histological/pathological alterations in the gonadal tissue of FS treated-mice compared with controls (data not shown). Given the robust effects of FS delivery, we next tested the potential for AAV1-FS delivered postnatally in a clinically meaningful paradigm to increase muscle mass and strength and delay muscle deterioration in the mdx mouse model of Duchenne muscular dystrophy (DMD). DMD is an X-linked recessive disease resulting in the wasting of skeletal muscles and cardiac function, ultimately resulting in death. Recently, FS was inves- tigated in mdx animals overexpressing a duplicated domain of the follistatin gene. Results demonstrated increased muscle mass and attenuated pathology, although the results were only doc- umented to 15 weeks of age (24). In our studies, mdx animals were injected bilaterally in the quadriceps and tibialis anterior muscles with a low (1 ϫ 10 10 viral particles) or high dose (1 ϫ 10 11 viral particles) of AAV1-FS at 3 weeks of age and followed for 5 months before necropsy. Increased levels of circulating FS were detected in the serum of both low and high dose treated animals with the high dose expressing the greatest levels of serum detected FS (high dose, 15.3 Ϯ 2.1 ng/ml; low dose, 6.8 Ϯ 0.4 ng/ml; GFP controls, 0 Ϯ 0.1 ng/ml; n ϭ 8 per group; P Ͻ 0.01). We demonstrated that AAV1-FS increased body mass compared with GFP treated controls, with the greatest increase in the high dose FS group (data not shown). Gross observation of AAV1-FS treated mice displayed a significant increase in muscle size compared with AAV1-GFP treated animals (Fig. 2 a ), with the greatest individual muscle weight increase in high dose FS-treated animals (Fig. 2 b ). Effects were not restricted to the injected muscles; they were also found at sites remote from directly targeted muscles (Fig. 2 b ). Increased muscle mass translated to a dose-dependent improvement in muscle strength in the hindlimbs and forelimbs of treated animals compared with GFP treated controls (Fig. 2 c ). Histological and morphometric analyses of AAV1-FS injected muscles and at remote sites demonstrated myofiber hypertrophy, supporting gross observations made at the time of necropsy (Fig. 3 a – c ). Furthermore, there was no shift in muscle fiber types in AAV-FS treated animals; however, there were fewer total fibers per square millimeter of area in the tibialis anterior muscle in animals treated with the high dose AAV-FS (Fig. 3 d and e ). Strikingly, FS-treated mice demonstrated a significant reduction in serum creatine kinase compared with GFP-treated controls (Fig. 4 a ). This is of interest, because FS was protective despite its lack of correction of the underlying dystrophin deficiency. The exact mechanism is not clear, but one might speculate that increasing the strength of individual fibers makes them less susceptible to damage from the stress of normal activities. The involvement of satellite cells in postnatal myostatin inhibition remains to be fully resolved; however, we did not see a statistical change in muscle satellite cell markers for FS-treated animals (data not shown). We also evaluated the potential for AAV1-FS to increase muscle strength in mdx animals when treated at an older age. We found that AAV1-FS injection at 210 days of age increased muscle strength Ϸ 60 days after administration and that the increased strength persisted long-term throughout the 560 days evaluated in this study (Fig. 4 b ). As early as 180 days of age, before AAV1-FS treatment, there was evident pathology in muscles of untreated mdx animals, with prominent endomysial connective tissue proliferation and inflammation (Fig. 4 c and d ). Pathological evaluation of gastrocnemius and diaphragm muscles at 560 days of age demonstrated that AAV1-FS treated animals had substantially fewer focal groups of necrotic muscle fibers and mononuclear cell infiltrates. Importantly, AAV1-FS treated animals had significantly reduced focal areas of endomysial connective tissue proliferation, which were pronounced in GFP treated animals, demonstrating that fibrosis, a hallmark of muscular dystrophy, was decreased in FS-treated animals (Fig. 4 c ). Pathology in the diaphragm also showed that FS-treatment reduced inflammation and fatty replacement compared with GFP-treated animals (Fig. 4 d ). Furthermore, AAV1-FS treatment demonstrated significant increases in muscle fiber diameters at this age compared with control ...
Context 2
... myostatin activity. Follistatin (FS) has been shown to bind to some TGF- ␤ family members and can function as a potent myostatin antagonist. Overexpression of follistatin by transgenic approaches in muscle has been shown to increase muscle growth in vivo (13), and a lack of follistatin results in reduced muscle mass at birth (14). Recent data has also shown that follistatin is capable of controlling muscle mass through pathways independent of the myostatin signaling cascade. In these studies, myostatin knockout mice were crossed to mice carrying a follistatin transgene. The resulting mice had a quadrupling of muscle mass compared with the doubling of muscle mass that is observed from lack of myostatin alone, confirming a role for follistatin in the regulation of muscle mass beyond solely myostatin inhibition (15). In addition to follistatin, two other proteins have been identified that are involved in the regulation of the myostatin. Follistatin-related gene (FLRG) is highly similar to follistatin and has been shown to inhibit activin and multiple bone mor- phogenic proteins in vitro (16, 17). Growth and differentiation factor-associated serum protein-1 (GASP-1) is a protein that has been discovered to contain multiple domains associated with protease-inhibitor proteins and a domain homologous to the 10-cysteine repeat found in follistatin. GASP-1 was shown to bind directly to the mature myostatin and myostatin propeptide and inhibits myostatin’s activity (18). Although recombinant protein injections or myostatin blocking antibodies are feasible strategies, gene therapy to express these myostatin inhibitor genes may prove a more efficacious therapeutic route for numerous reasons, including the lack of potential immune response to antibody treatment and the requirement for multiple injections. Here, we report that a one-time postnatal intramuscular injection of adeno-associated virus (AAV) encoding myostatin- inhibitor-proteins resulted in long-term improvement of muscle size and strength in wild-type animals. Delivery of a myostatin- inhibitor-protein in dystrophic mdx animals reversed muscle pathology and improved strength, even when administered in 6.5-month-old animals. Specifically, we show here that follista- tin-344 resulted in the greatest effects on muscle size and function and was well tolerated with no untoward effects on cardiac pathology or reproductive capacity in either male or female treated animals. AAV-mediated gene delivery to muscle provides a system to generate high levels of protein in the target tissue or by a secreted product carried to remote sites through the circulation (19). We cloned the known secreted myostatin-inhibiting genes, including growth and differentiation factor-associated serum protein-1 (GASP-1) (18), follistatin-related gene (FLRG) (17), and follista- tin-344 (FS) (13) into AAV serotype 1, which have demonstrated high muscle transduction capabilities. There are two isoforms of follistatin generated by alternative splicing. The FS-344 variant undergoes peptide cleavage to generate the FS-315 isoform and the other FS-317 variant produces the FS-288 isoform after peptide cleavage. We used the human FS-344 variant, which exclusively generates the serum circulating FS-315 isoform of FS and includes a C-terminal acidic region (20). We chose FS-344 (FS), because the other FS-317 isoform, lacking the C terminus, shows preferential localization to the ovarian follicular fluid and high tissue binding affinity through heparin sulfate proteoglycans, which may affect reproductive capacity and bind to other off-target sites (21). FS-288 represents the membrane-bound form of follistatin (22), is a potent suppressor of pituitary follicle stimulating hormone (23), is found in the follicular fluid of the ovary and in the testes, and demonstrates a high affinity for the granulosa cells of the ovary. We sought to determine the efficacy of these proteins to increase muscle mass in normal and dystrophic mice. We administered 1 ϫ 10 11 AAV1 viral particles per animal encoding FS, FLRG, GASP-1, or GFP bilaterally into the quadriceps and tibialis anterior muscles of 4-week-old wild-type C57BL/6 mice. All animals treated with the myostatin inhibitors demonstrated an increase in body mass with an observable gross enhancement of muscles when analyzed at 725-days of age compared with GFP-treated controls (Fig. 1 a and b ). Evaluation of individual muscle weights showed an increase in muscle mass for all myostatin inhibitor-treated animals, with the greatest increase in FS-treated animals. The increased muscle mass was found in the injected hindlimb muscles and remote muscles to the injection site, such as the triceps. Thus, these inhibitors were secreted into the circulation from the site of muscle injection, enhancing skeletal muscle mass at remote sites (Fig. 1 c ). The enlarged muscle mass was accompanied by functional improvement demonstrated by an increase in hindlimb grip strength (Fig. 1 d ). There was no effect on heart mass or histological appearance of cardiomyocytes, indicating that myostatin inhibition was selec- tive to skeletal muscle tissue (data not shown). There has been concern that FS adversely effects gonadal function. We found no change in reproductive capacity in mice treated with our AAV1 carrying the FS344 transgene (AAV1-FS, Table 1) Furthermore, we found no histological/pathological alterations in the gonadal tissue of FS treated-mice compared with controls (data not shown). Given the robust effects of FS delivery, we next tested the potential for AAV1-FS delivered postnatally in a clinically meaningful paradigm to increase muscle mass and strength and delay muscle deterioration in the mdx mouse model of Duchenne muscular dystrophy (DMD). DMD is an X-linked recessive disease resulting in the wasting of skeletal muscles and cardiac function, ultimately resulting in death. Recently, FS was inves- tigated in mdx animals overexpressing a duplicated domain of the follistatin gene. Results demonstrated increased muscle mass and attenuated pathology, although the results were only doc- umented to 15 weeks of age (24). In our studies, mdx animals were injected bilaterally in the quadriceps and tibialis anterior muscles with a low (1 ϫ 10 10 viral particles) or high dose (1 ϫ 10 11 viral particles) of AAV1-FS at 3 weeks of age and followed for 5 months before necropsy. Increased levels of circulating FS were detected in the serum of both low and high dose treated animals with the high dose expressing the greatest levels of serum detected FS (high dose, 15.3 Ϯ 2.1 ng/ml; low dose, 6.8 Ϯ 0.4 ng/ml; GFP controls, 0 Ϯ 0.1 ng/ml; n ϭ 8 per group; P Ͻ 0.01). We demonstrated that AAV1-FS increased body mass compared with GFP treated controls, with the greatest increase in the high dose FS group (data not shown). Gross observation of AAV1-FS treated mice displayed a significant increase in muscle size compared with AAV1-GFP treated animals (Fig. 2 a ), with the greatest individual muscle weight increase in high dose FS-treated animals (Fig. 2 b ). Effects were not restricted to the injected muscles; they were also found at sites remote from directly targeted muscles (Fig. 2 b ). Increased muscle mass translated to a dose-dependent improvement in muscle strength in the hindlimbs and forelimbs of treated animals compared with GFP treated controls (Fig. 2 c ). Histological and morphometric analyses of AAV1-FS injected muscles and at remote sites demonstrated myofiber hypertrophy, supporting gross observations made at the time of necropsy (Fig. 3 a – c ). Furthermore, there was no shift in muscle fiber types in AAV-FS treated animals; however, there were fewer total fibers per square millimeter of area in the tibialis anterior muscle in animals treated with the high dose AAV-FS (Fig. 3 d and e ). Strikingly, FS-treated mice demonstrated a significant reduction in serum creatine kinase compared with GFP-treated controls (Fig. 4 a ). This is of interest, because FS was protective despite its lack of correction of the underlying dystrophin deficiency. The exact mechanism is not clear, but one might speculate that increasing the strength of individual fibers makes them less susceptible to damage from the stress of normal activities. The involvement of satellite cells in postnatal myostatin inhibition remains to be fully resolved; however, we did not see a statistical change in muscle satellite cell markers for FS-treated animals (data not shown). We also evaluated the potential for AAV1-FS to increase muscle strength in mdx animals when treated at an older age. We found that AAV1-FS injection at 210 days of age increased muscle strength Ϸ 60 days after administration and that the increased strength persisted long-term throughout the 560 days evaluated in this study (Fig. 4 b ). As early as 180 days of age, before AAV1-FS treatment, there was evident pathology in muscles of untreated mdx animals, with prominent endomysial connective tissue proliferation and inflammation (Fig. 4 c and d ). Pathological evaluation of gastrocnemius and diaphragm muscles at 560 days of age demonstrated that AAV1-FS treated animals had substantially fewer focal groups of necrotic muscle fibers and mononuclear cell infiltrates. Importantly, AAV1-FS treated animals had significantly reduced focal areas of endomysial connective tissue proliferation, which were pronounced in GFP treated animals, demonstrating that fibrosis, a hallmark of muscular dystrophy, was decreased in FS-treated animals (Fig. 4 c ). Pathology in the diaphragm also showed that FS-treatment reduced inflammation and fatty replacement compared with GFP-treated animals (Fig. 4 d ). Furthermore, AAV1-FS treatment demonstrated significant increases in muscle fiber diameters at this age compared with control GFP-treated animals (Fig. 4 c and d ). These results demonstrated that myostatin inhibition by FS treatment was beneficial in aged mdx ...
Context 3
... dystrophy mice treated with deacetylase inhibitors. The resulting muscle enhancement was attributed to an increase in the protein follistatin, which has been shown in part to inhibit the activity of myostatin (12). Trichostatin A (TSA) treatment required daily administration and was not evaluated in aged animals where off target effects may exist. The identification of myostatin binding proteins capable of regulating myostatin activity has led to potential new approaches for postnatal muscle enhancement and expanded the potential for gene therapy to be considered as a method to inhibit myostatin activity. Follistatin (FS) has been shown to bind to some TGF- ␤ family members and can function as a potent myostatin antagonist. Overexpression of follistatin by transgenic approaches in muscle has been shown to increase muscle growth in vivo (13), and a lack of follistatin results in reduced muscle mass at birth (14). Recent data has also shown that follistatin is capable of controlling muscle mass through pathways independent of the myostatin signaling cascade. In these studies, myostatin knockout mice were crossed to mice carrying a follistatin transgene. The resulting mice had a quadrupling of muscle mass compared with the doubling of muscle mass that is observed from lack of myostatin alone, confirming a role for follistatin in the regulation of muscle mass beyond solely myostatin inhibition (15). In addition to follistatin, two other proteins have been identified that are involved in the regulation of the myostatin. Follistatin-related gene (FLRG) is highly similar to follistatin and has been shown to inhibit activin and multiple bone mor- phogenic proteins in vitro (16, 17). Growth and differentiation factor-associated serum protein-1 (GASP-1) is a protein that has been discovered to contain multiple domains associated with protease-inhibitor proteins and a domain homologous to the 10-cysteine repeat found in follistatin. GASP-1 was shown to bind directly to the mature myostatin and myostatin propeptide and inhibits myostatin’s activity (18). Although recombinant protein injections or myostatin blocking antibodies are feasible strategies, gene therapy to express these myostatin inhibitor genes may prove a more efficacious therapeutic route for numerous reasons, including the lack of potential immune response to antibody treatment and the requirement for multiple injections. Here, we report that a one-time postnatal intramuscular injection of adeno-associated virus (AAV) encoding myostatin- inhibitor-proteins resulted in long-term improvement of muscle size and strength in wild-type animals. Delivery of a myostatin- inhibitor-protein in dystrophic mdx animals reversed muscle pathology and improved strength, even when administered in 6.5-month-old animals. Specifically, we show here that follista- tin-344 resulted in the greatest effects on muscle size and function and was well tolerated with no untoward effects on cardiac pathology or reproductive capacity in either male or female treated animals. AAV-mediated gene delivery to muscle provides a system to generate high levels of protein in the target tissue or by a secreted product carried to remote sites through the circulation (19). We cloned the known secreted myostatin-inhibiting genes, including growth and differentiation factor-associated serum protein-1 (GASP-1) (18), follistatin-related gene (FLRG) (17), and follista- tin-344 (FS) (13) into AAV serotype 1, which have demonstrated high muscle transduction capabilities. There are two isoforms of follistatin generated by alternative splicing. The FS-344 variant undergoes peptide cleavage to generate the FS-315 isoform and the other FS-317 variant produces the FS-288 isoform after peptide cleavage. We used the human FS-344 variant, which exclusively generates the serum circulating FS-315 isoform of FS and includes a C-terminal acidic region (20). We chose FS-344 (FS), because the other FS-317 isoform, lacking the C terminus, shows preferential localization to the ovarian follicular fluid and high tissue binding affinity through heparin sulfate proteoglycans, which may affect reproductive capacity and bind to other off-target sites (21). FS-288 represents the membrane-bound form of follistatin (22), is a potent suppressor of pituitary follicle stimulating hormone (23), is found in the follicular fluid of the ovary and in the testes, and demonstrates a high affinity for the granulosa cells of the ovary. We sought to determine the efficacy of these proteins to increase muscle mass in normal and dystrophic mice. We administered 1 ϫ 10 11 AAV1 viral particles per animal encoding FS, FLRG, GASP-1, or GFP bilaterally into the quadriceps and tibialis anterior muscles of 4-week-old wild-type C57BL/6 mice. All animals treated with the myostatin inhibitors demonstrated an increase in body mass with an observable gross enhancement of muscles when analyzed at 725-days of age compared with GFP-treated controls (Fig. 1 a and b ). Evaluation of individual muscle weights showed an increase in muscle mass for all myostatin inhibitor-treated animals, with the greatest increase in FS-treated animals. The increased muscle mass was found in the injected hindlimb muscles and remote muscles to the injection site, such as the triceps. Thus, these inhibitors were secreted into the circulation from the site of muscle injection, enhancing skeletal muscle mass at remote sites (Fig. 1 c ). The enlarged muscle mass was accompanied by functional improvement demonstrated by an increase in hindlimb grip strength (Fig. 1 d ). There was no effect on heart mass or histological appearance of cardiomyocytes, indicating that myostatin inhibition was selec- tive to skeletal muscle tissue (data not shown). There has been concern that FS adversely effects gonadal function. We found no change in reproductive capacity in mice treated with our AAV1 carrying the FS344 transgene (AAV1-FS, Table 1) Furthermore, we found no histological/pathological alterations in the gonadal tissue of FS treated-mice compared with controls (data not shown). Given the robust effects of FS delivery, we next tested the potential for AAV1-FS delivered postnatally in a clinically meaningful paradigm to increase muscle mass and strength and delay muscle deterioration in the mdx mouse model of Duchenne muscular dystrophy (DMD). DMD is an X-linked recessive disease resulting in the wasting of skeletal muscles and cardiac function, ultimately resulting in death. Recently, FS was inves- tigated in mdx animals overexpressing a duplicated domain of the follistatin gene. Results demonstrated increased muscle mass and attenuated pathology, although the results were only doc- umented to 15 weeks of age (24). In our studies, mdx animals were injected bilaterally in the quadriceps and tibialis anterior muscles with a low (1 ϫ 10 10 viral particles) or high dose (1 ϫ 10 11 viral particles) of AAV1-FS at 3 weeks of age and followed for 5 months before necropsy. Increased levels of circulating FS were detected in the serum of both low and high dose treated animals with the high dose expressing the greatest levels of serum detected FS (high dose, 15.3 Ϯ 2.1 ng/ml; low dose, 6.8 Ϯ 0.4 ng/ml; GFP controls, 0 Ϯ 0.1 ng/ml; n ϭ 8 per group; P Ͻ 0.01). We demonstrated that AAV1-FS increased body mass compared with GFP treated controls, with the greatest increase in the high dose FS group (data not shown). Gross observation of AAV1-FS treated mice displayed a significant increase in muscle size compared with AAV1-GFP treated animals (Fig. 2 a ), with the greatest individual muscle weight increase in high dose FS-treated animals (Fig. 2 b ). Effects were not restricted to the injected muscles; they were also found at sites remote from directly targeted muscles (Fig. 2 b ). Increased muscle mass translated to a dose-dependent improvement in muscle strength in the hindlimbs and forelimbs of treated animals compared with GFP treated controls (Fig. 2 c ). Histological and morphometric analyses of AAV1-FS injected muscles and at remote sites demonstrated myofiber hypertrophy, supporting gross observations made at the time of necropsy (Fig. 3 a – c ). Furthermore, there was no shift in muscle fiber types in AAV-FS treated animals; however, there were fewer total fibers per square millimeter of area in the tibialis anterior muscle in animals treated with the high dose AAV-FS (Fig. 3 d and e ). Strikingly, FS-treated mice demonstrated a significant reduction in serum creatine kinase compared with GFP-treated controls (Fig. 4 a ). This is of interest, because FS was protective despite its lack of correction of the underlying dystrophin deficiency. The exact mechanism is not clear, but one might speculate that increasing the strength of individual fibers makes them less susceptible to damage from the stress of normal activities. The involvement of satellite cells in postnatal myostatin inhibition remains to be fully resolved; however, we did not see a statistical change in muscle satellite cell markers for FS-treated animals (data not shown). We also evaluated the potential for AAV1-FS to increase muscle strength in mdx animals when treated at an older age. We found that AAV1-FS injection at 210 days of age increased muscle strength Ϸ 60 days after administration and that the increased strength persisted long-term throughout the 560 days evaluated in this study (Fig. 4 b ). As early as 180 days of age, before AAV1-FS treatment, there was evident pathology in muscles of untreated mdx animals, with prominent endomysial connective tissue proliferation and inflammation (Fig. 4 c and d ). Pathological evaluation of gastrocnemius and diaphragm muscles at 560 days of age demonstrated that AAV1-FS treated animals had substantially fewer focal groups of necrotic muscle fibers and mononuclear cell infiltrates. Importantly, AAV1-FS treated animals had significantly reduced focal areas of endomysial ...

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... Activin and myostatin are growth factors that belong to the TGF-β superfamily, and they are known to have negative effects on muscle and bone mass [108,109]; therefore, their downregulation might be considered a therapeutic approach to prevent muscle wasting and bone degeneration in DMD patients [71]. Follistatin modulates bone metabolism presumably via activin and myostatin signaling, and follistatin-based gene therapy was shown to have positive effects on muscles [110][111][112]. However, myostatin seems to have a positive impact on tendons. ...
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Duchenne muscular dystrophy (DMD) is a devastating chromosome X-linked disease that manifests predominantly in progressive skeletal muscle wasting and dysfunctions in the heart and diaphragm. Approximately 1/5000 boys and 1/50,000,000 girls suffer from DMD, and to date, the disease is incurable and leads to premature death. This phenotypic severity is due to mutations in the DMD gene, which result in the absence of functional dystrophin protein. Initially, dystrophin was thought to be a force transducer; however, it is now considered an essential component of the dystrophin-associated protein complex (DAPC), viewed as a multicomponent mechanical scaffold and a signal transduction hub. Modulating signal pathway activation or gene expression through epigenetic modifications has emerged at the forefront of therapeutic approaches as either an adjunct or stand-alone strategy. In this review, we propose a broader perspective by considering DMD to be a disease that affects myofibers and muscle stem (satellite) cells, as well as a disorder in which abrogated communication between different cell types occurs. We believe that by taking this systemic view, we can achieve safe and holistic treatments that can restore correct signal transmission and gene expression in diseased DMD tissues.
... Subsequently, it was reported that FST-induced muscle hypertrophy was associated inhibition of both MST and activin A and induction of satellite cell proliferation (137). Fst gene delivery of AAV1-FST344 in normal and dystrophic mice as well as in non-human primates led to significant increase in muscle mass and strength (138,139). Transgenic expression of Fst in mdx mice, a popular model for Duchenne muscular dystrophy (DMD), showed amelioration of dystrophic pathology and increase in skeletal muscle mass (140). Interestingly, in a gene therapy trial Mendell et al. demonstrated beneficial effects of FST344 direct delivery into intramuscular quadriceps in patients suffering from Becker Muscular Dystrophy without any apparent side effects (141). ...
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Obesity is a global health problem and a major risk factor for several metabolic conditions including dyslipidemia, diabetes, insulin resistance and cardiovascular diseases. Obesity develops from chronic imbalance between energy intake and energy expenditure. Stimulation of cellular energy burning process has the potential to dissipate excess calories in the form of heat via the activation of uncoupling protein-1 (UCP1) in white and brown adipose tissues. Recent studies have shown that activation of transforming growth factor-β (TGF-β) signaling pathway significantly contributes to the development of obesity, and blockade or inhibition is reported to protect from obesity by promoting white adipose browning and increasing mitochondrial biogenesis. Identification of novel compounds that activate beige/brown adipose characteristics to burn surplus calories and reduce excess storage of fat are actively sought in the fight against obesity. In this review, we present recent developments in our understanding of key modulators of TGF-β signaling pathways including follistatin (FST) and myostatin (MST) in regulating adipose browning and brown adipose mass and activity. While MST is a key ligand for TGF-β family, FST can bind and regulate biological activity of several TGF-β superfamily members including activins, bone morphogenic proteins (BMP) and inhibins. Here, we review the literature supporting the critical roles for FST, MST and other proteins in modulating TGF-β signaling to influence beige and brown adipose characteristics. We further review the potential therapeutic utility of FST for the treatment of obesity and related metabolic disorders.
... Follistatin, a natural antagonist of myostatin, was overexpressed via delivery with adeno-associated virus sub-type 9 (AAV9). This strategy of myostatin inhibition has been shown to result in increased muscle mass in young rodents and nonhuman primates ( Haidet et al., 2008 ;Kota et al., 2009 ;Rodino-Klapac et al., 2009 ). Normal development and maintenance of mo-tor unit and NMJ function are dependent on bidirectional trophic interactions between muscle and motor neurons ( Delbono, 2003 ;Messi and Delbono, 2003 ). ...
... Mice were then randomized, stratified by sex, and separated into individual standard cages (single-housed, one mouse per cage) fitted with voluntary running wheels with a diameter of 10.16 cm (Columbus Instruments, USA) (n = 36, 18 males, 18 females) or without voluntary running wheels (n = 14, 7 males, 7 females). Of the 36 mice that were randomized into cages with wheels, 16 mice (8 male, 8 female) were also randomized to treatment with intramuscular injection of AAV9-FS344 (1.13 × 10 11 vg/mouse) bilaterally in the gastrocnemius and tibialis anterior muscles to overexpress a human isoform of follistatin, FS344, (FST) at baseline ( Haidet et al., 2008 ). Mice that did not receive FST injection received injections with vehicle (saline). ...
Article
Sarcopenia, or pathological loss of muscle mass and strength during aging, is an important contributor to loss of physical function in older adults. Sarcopenia is a multifactorial syndrome associated with intrinsic muscle and upstream neurological dysfunction. Exercise is well-established as an effective intervention for sarcopenia, but less is known about the long-term neurobiological impact of exercise. The goals of this study were to investigate the effects of exercise, alone or in combination with follistatin (FST) overexpression (antagonist of myostatin), on neuromuscular junction transmission and motor unit numbers in mice between the age of 22 and 27 months, ages at which prior studies have demonstrated that some motor unit loss is already evident. C57BL/6J mice underwent baseline assessment and were randomized to housing with or without voluntary running wheels and injection with adeno-associated virus to overexpress FST or vehicle. Groups for comparison included sedentary and running with and without FST. Longitudinal assessments showed significantly increased muscle mass and contractility in the ‘running plus FST’ group, but running, with and without FST, showed no effect on motor unit degeneration. In contrast, running, with and without FST, demonstrated marked improvement of neuromuscular junction transmission stability.
... Myostatin, another Transforming Growth Factor-β (TGFβ) family member with established cachectic and profibrotic properties [23], is produced by muscles and inhibits muscle growth by acting through either activin-like kinase 4 (Alk 4) or activin-like kinase 5 (Alk 5) and the activin type II receptors [24] This indicates that myostatin shares a signaling pathway with the activins and that follistatin also binds, albeit less effectively, to myostatin. The ability of follistatin to prevent muscle wasting in cachexia and fibrosis in mouse models of muscular dystrophy probably involves interference with myostatin signaling via the pathway, although the relative contributions of endogenous activin and myostatin have not been well characterized in these models [25][26][27]. ...
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Sarcopenia is characterized by progressive and generalized loss of skeletal muscle mass and strength that occurs with aging or in association with various diseases. The condition is prevalent worldwide and occurs more frequently in patients with chronic diseases owing to the intrinsic relationship of muscles with glucose, lipid, and protein metabolism. Liver cirrhosis is characterized by the progression of necro-inflammatory liver diseases, which leads to fibrosis, portal hypertension, and a catabolic state, which causes loss of muscle tissue. Sarcopenia is of significant concern in the state of liver cirrhosis because sarcopenia has been associated with higher mortality, increased hospital admissions, worse post-liver transplant outcomes, decreased quality of life, and increased risk for other complications associated with cirrhosis. Therefore, sarcopenia is also an important feature of liver cirrhosis, representing a negative prognostic factor and influencing mortality. An increased understanding of sarcopenia could lead to the development of novel therapeutic approaches that could help improve the cognitive impairment of cirrhotic patients; therefore, we present a review of the mechanisms and diagnosis of sarcopenia in liver disease and existing therapeutic approaches.
... Targeting the follistatin-myostatin pathway by either direct myostatin blockade or follistatin overexpression has revealed valuable therapeutic potential in the treatment of muscular dystrophies (5)(6)(7)(8). Thus, the understanding of the mechanism by which compounds of pharmacological interest, such as DIs and NO donors, target the follistatin-myostatin pathway is instrumental to develop effective strategies in the treatment of muscular dystrophies. ...
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The overlapping histological and biochemical features underlying the beneficial effect of deacetylase inhibitors and NO donors in dystrophic muscles suggest an unanticipated molecular link among dystrophin, NO signaling, and the histone deacetylases (HDACs). Higher global deacetylase activity and selective increased expression of the class I histone deacetylase HDAC2 were detected in muscles of dystrophin-deficient MDX mice. In vitro and in vivo siRNA-mediated down-regulation of HDAC2 in dystrophic muscles was sufficient to replicate the morphological and functional benefits observed with deacetylase inhibitors and NO donors. We found that restoration of NO signaling in vivo, by adenoviral-mediated expression of a constitutively active endothelial NOS mutant in MDX muscles, and in vitro, by exposing MDX-derived satellite cells to NO donors, resulted in HDAC2 blockade by cysteine S-nitrosylation. These data reveal a special contribution of HDAC2 in the pathogenesis of Duchenne muscular dystrophy and indicate that HDAC2 inhibition by NO-dependent S-nitrosylation is important for the therapeutic response to NO donors in MDX mice. They also define a common target for independent pharmacological interventions in the treatment of Duchenne muscular dystrophy. epigenetic ͉ skeletal muscle R ecent studies performed in mouse models of muscular dystrophy have reported on the therapeutic potential of pharmacological interventions that target events downstream to the genetic defect responsible for the disease. For instance, histone deacetylase inhibitors (DIs) and NO donors countered the progression of muscular dystrophy in MDX mice (1, 2). Intriguingly, the beneficial effect of both treatments relies, at least in part, on the formation of myofibers larger than normal and on the transcriptional activation of the myostatin antagonist follistatin (1, 3, 4). This evidence suggests a common mechanism linking the effect of NO donors and that of DIs in dystrophic muscles. Targeting the follistatin-myostatin pathway by either direct myostatin blockade or follistatin overexpression has revealed valuable therapeutic potential in the treatment of muscular dystrophies (5-8). Thus, the understanding of the mechanism by which compounds of pharmacological interest, such as DIs and NO donors, target the follistatin-myostatin pathway is instrumental to develop effective strategies in the treatment of muscular dystrophies. Follistatin expression in skeletal muscles is regulated by class I histone deacetylases (HDACs) (1, 4), which inhibit the activity of MyoD (9, 10) and additional transcription factors, such as CREB and NFAT, possibly recruited to the follistatin promoter (4). In the present study we have investigated the individual role of class I HDACs in the progression of muscular dystrophy in MDX mice and evaluated the possibility that DIs and NO signaling could converge on HDAC blockade. We show that class I HDAC2 expression and activity are increased in skeletal muscle from MDX mice. HDAC2 down-regulation by siRNA enhances the ability of MDX derived-satellite cells to form multinucleated myotubes in vitro and is sufficient to counter the disease progression in vivo, when delivered to MDX muscles. Finally, we show that HDAC2 S-nitrosylation by NO donors impairs its enzymatic function. These results indicate that HDAC2 is an important common pharmacological target of distinct pharmacological interventions aimed at Duchenne muscular dystrophy (DMD) treatment and suggest a novel mechanism of HDAC2 inhibition by NO-dependent cysteine S-nitrosylation.
... In addition, rAAV-mediated delivery of alternative candidates that could ameliorate DMD-associated phenotypes, such as follistatin (FST; CDS 954 bp) and B4GALNT2 (β-1, 4-N-acetylgalactosaminyltransferase 2; CDS 1443 bp), has been under clinical trials for the past few years 44,45 , but with limited efficacy. FST is a myostatin inhibitor, and its overexpression increases muscle mass and strength in the DMD mdx mouse model 46 . Of note, the phenotype of this mouse model differs from human DMD, having reduced endomysial fibrosis and successful fibre regeneration 47 . ...
Article
Over a thousand diseases are caused by mutations that alter gene expression levels. The potential of nuclease-deficient zinc fingers, TALEs or CRISPR fusion systems to treat these diseases by modulating gene expression has recently emerged. These systems can be applied to modify the activity of gene-regulatory elements - promoters, enhancers, silencers and insulators, subsequently changing their target gene expression levels to achieve therapeutic benefits - an approach termed cis-regulation therapy (CRT). Here, we review emerging CRT technologies and assess their therapeutic potential for treating a wide range of diseases caused by abnormal gene dosage. The challenges facing the translation of CRT into the clinic are discussed.
... Upon treatment, patients were reported to have reduced appetite and improved behaviour [97,98]. For hypotonia, a myostatin inhibitor may promote muscle growth and tone, and may improve metabolism [99] for hyperphagia/ obesity. There are a number of other experimental interventions to treat hyperphagia/obesity as well [98,[100][101][102][103][104]. ...
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Prader-Willi syndrome (PWS) is caused by the loss of function of the paternally inherited 15q11-q13 locus. This region is governed by genomic imprinting, a phenomenon in which genes are expressed exclusively from one parental allele. The genomic imprinting of the 15q11-q13 locus is established in the germline and is largely controlled by a bipartite imprinting centre. One part, termed the Prader-Willi syndrome imprinting center (PWS-IC), comprises a CpG island that is unmethylated on the paternal allele and methylated on the maternal allele. The second part, termed the Angelman syndrome imprinting centre, is required to silence the PWS_IC in the maternal germline. The loss of the paternal contribution of the imprinted 15q11-q13 locus most frequently occurs owing to a large deletion of the entire imprinted region but can also occur through maternal uniparental disomy or an imprinting defect. While PWS is considered a contiguous gene syndrome based on large-deletion and uniparental disomy patients, the lack of expression of only non-coding RNA transcripts from the SNURF-SNRPN/SNHG14 may be the primary cause of PWS. Patients with small atypical deletions of the paternal SNORD116 cluster alone appear to have most of the PWS related clinical phenotypes. The loss of the maternal contribution of the 15q11-q13 locus causes a separate and distinct condition called Angelman syndrome. Importantly, while much has been learned about the regulation and expression of genes and transcripts deriving from the 15q11-q13 locus, there remains much to be learned about how these genes and transcripts contribute at the molecular level to the clinical traits and developmental aspects of PWS that have been observed.