Bogdanovich et al. were the first to successfully treat the commonly used mouse model of Duchenne Muscular Dystrophy (DMD), the mdx, with antibodies directed towards myostatin (monoclonal antibodies, JA16) [73]. The results were promising, as the diaphragm and the skeletal muscle, which in the mdx reproduces the pathological features seen in muscles of DMD-patients most accurately [132], showed fewer degenerative features compared to controls. Meanwhile, m. extensor digitorum longus (EDL) had increased weight, cross-sectional area (CSA) and absolute force but failed to show improvement on specific force and stretch resistance. Similar results with increased muscle weight and absolute force but lack of improvement in specific force and resistance were seen in the Sgcg^−/− model of limb-girdle muscular dystrophy (LGMD) 2C in a design of similar age and treatment length to the previously mentioned study [74]. The Sgcd^−/− mouse model of LGMD2F also treated with JA16 antibodies was not able to improve fibrosis in either young or older Sgcd^−/− animals (4 and 20 weeks old at treatment start, respectively) with older animals even showing signs of worsening of fibrosis [75]. Interestingly, a 5-week treatment period of very young (16 days old) mdx-animals showed positive effects on the diaphragm, as specific force increased while absolute force was unaffected, fiber size increased and connective tissue infiltration of the diaphragm was reduced [76], indicating that early initiation of treatment is crucial for a positive effect. Another monoclonal antibody developed by Pfizer, mRK-35, was also able to increase absolute but not specific force in mdx mice [72] and the TgActa1^D286G mouse model of nemaline myopathy [78]. Treatment of the Sod1^G93A mouse and rat models of amyotrophic lateral sclerosis (ALS) with RK35 improved grip strength compared to placebo controls but did not delay disease onset or extend survival of either model [80]. Later, Muramatsu and colleagues introduced the concept of “sweeping antibody technology” with the GYM329-antibody designed to bind and clear latent myostatin from the circulatory system, which increased muscle mass in three mice models and cynomolgus monkeys and also improved grip strength in the mice [71]. As opposed to other antimyostatin antibodies, GYM329 did not bind GDF-11 and this specificity appears to induce an enhanced effect on muscle mass in treated animals. Especially in older animals, where other myostatin inhibition treatments fail or struggle to achieve an effect, GYM329 appeared superior. Other models of neuromuscular disorders such as the SmnΔ7 mouse of spinal muscle atrophy (SMA) had increased absolute muscle torque but not specific torque after treatment with the muSRK-015P antibody versus myostatin (combined with salvation of Smn2-gene mRNA) [133]. In a study of micro-gravity-induced muscular atrophy, mice were held at the International Space Station and treated with YN41 for 6 weeks, inducing improved grip strength compared to controls, as well as increased muscle mass [68].

As previously mentioned, the myostatin propeptide functions as an inhibitor of myostatin, as it binds myostatin in an inactive complex. Propeptide-based inhibition by intraperitoneal injection for three months resulted in increased body mass, EDL mass, absolute and specific force in EDL. There was no effect on stretch-resistance but the histopathological phenotype of the diaphragm improved compared to untreated mdx [84]. Bartoli et al. treated calpain-3-null and Sgca^−/−-mouse models of LGMD2A and 2D, respectively, by local and systemic overexpression of the propeptide but were only able to improve the calpain-3-null mice [86].

In order to increase the specific targeting of myostatin and reduce binding of the variety of other ligands that also bind to and activate ActRIIB, another approach based on the systemic administration of a soluble activin type IIB receptor, sActRIIB, has been widely utilized (Supplementary Table S1). The compound RAP-031, developed by Acceleron, is a fusion protein consisting of the extracellular domain of the ActRIIB linked to the Fc-portion of murine IgG to delay systemic clearance. Applying this approach, Pistilli and colleagues demonstrated an increase in both absolute and specific force of EDL in mdx mice [98], a functional outcome, which unfortunately has been difficult to replicate in both wild-type [87,93], mdx [92,94,99], and nemaline myopathy mouse models [101] (Supplementary Table S1). The treatment of mice with muscular atrophy due to spinal cord injury with RAP-031 did not alleviate the atrophy [134]. A hypoxia model in wild-type mice showed improved resistance to eccentric lengthening but no other studies using the soluble receptor have shown improvements to stretch resistance [135]. The specific hypertrophy of fibers with a IIB fiber-type composition was observed in two models of myotubularin-deficient mice [90,102] but also in other fibers of wild-type animals [89,90].

Blocking the ActRIIB itself by antibodies has not been widely used as another means of myostatin inhibition. Novartis developed BYM338 (bimagrumab, which would progress into clinical trials as mentioned below) and described the receptor-specificity in cell cultures and myoblasts while also showing the effects on body and muscle mass in both SCID-mice and a glucocorticoid atrophy model [106].

Like the myostatin propeptide, follistatin is able to inhibit not only myostatin but also shows affinity for other TGF-β-family members (such as BMPs and activins) [24,136]. Transgenic overexpression of follistatin primarily showed increased muscle weight and fiber diameter [19]. Transgenic mice overexpressing a follistatin-derived myostatin inhibitor crossed with the mdx ameliorated the dystrophic features in terms of grip strength and pathohistological features [137]. When transgenic overexpression of follistatin (F66-mice) is crossed with the dysferlinopathy LGMD2B model Dysf^−/−, the positive effect on muscle weight in F66;Dysf^−/−-mice declines with age and the specific force of EDL is reduced, compared to F66-mice, exacerbating the dystrophic phenotype [104]. Furthermore, ActRIIB-FC-administration in Dysf^−/−-mice ameliorated histopathological changes, but increased creatine kinase (CK, a marker of muscular damage and membrane integrity) levels. The authors conclude that follistatin overexpression accelerated the degenerative features in the dysferlinopathy model, as the dystrophin-deficient mdx was not exacerbated [104], and suggest that muscle hypertrophy may have pernicious effects depending on the disease context.

Another approach using a follistatin-based fusion protein ACE-083 by local intramuscular injections increased CSA, weight and absolute, but not specific, force of injected muscle tibialis anterior (TA) in the Trembler-J mouse model of Charcot–Marie–Tooth disease and mdx [111].

As the systemic clearance of follistatin is rather quick, systemic versus local administration poses a challenge. Thus, the pharmacokinetic properties were edited and a long-acting follistatin-based molecule (FS-EEE-hFc) was engineered by Shen and colleagues [138] and applied by intravenous and subcutaneous administration to wild-type and mdx-animals [99]. The subcutaneous treatment of young (4 weeks) mdx-mice for 12 weeks also undergoing an exhaustion-exercise regime showed increased muscle weight and absolute but not specific force increments [99].

Using the same approach as mentioned earlier with adeno-associated virus 8 (AAV8)-delivered myostatin inhibitors, Morine and colleagues treated the mdx with AAV-vectors, which brought liver-mediated transcripts of sActRIIB [115] or dnMSTN [116] into circulation. The sActRIIB treatment did increase the muscle mass, fiber size and absolute force of the EDL, while CK decreased. However, there were no positive effects in soleus or specific force [115]. The dnMSTN paper showed that the treatment in mdx-mice was predominantly observed in the fast fibers (IIA, IIX and IIB) of both the EDL and soleus, while soleus increased both absolute and specific force and CK decreased [116]. A similar study in the D2.mdx only reported beneficial effects on absolute force in EDL [117].

Similar to the treatment regimes in the mdx, Liu and colleagues treated the C/C mouse model of spinal muscle atrophy (SMA) with AAV8-vectors containing transcripts for dnMSTN and sActRIIB, respectively [114]. Both treatments increased the size of type IIA and IIB-fibers, leaving type I-fibers unaffected (IIX was not measured). While specific force was unaffected by treatment, absolute force increased in EDL (both treatments) and soleus (only sActRIIB).

Another approach was used in wild-type MF-1 mice, where propeptide coupled to an immunoglobulin Fc molecule was delivered by means of AAV8 vectors to hepatocytes, ensuring an intrinsic production of the inhibitor [113]. In contrast to exogenic injections of the propeptide, Foster and colleagues treated mice from six weeks of age and found an increased absolute force in oxidative muscle soleus but not in EDL. Both EDL and soleus increased CSA, as well as subanalyses of fiber-types I, IIA and IIB. In a similar design, mdx-mice were treated at the age of three months, which increased body mass, grip strength, muscle mass and fiber radius [85]. The absolute twitch and tetanic force production improved but specific force did not.

Myostatin has also been sought downregulated by means of RNA interference. Dumonceaux et al. combined short hairpin RNA (shRNA) interference of ActRIIB mRNA with AAV mediated exon-skipping of dystrophin. The number of fibers increased in TA, but force production was unchanged in mice that received myostatin interference solely compared to untreated mdx [120].

In contrast to AAV-mediated gene therapy, antisense oligomers (AOs) hold no risk of uncontrolled genome insertion and levels of exon skipping can be regulated or aborted over time. Antisense phosphorodiamidate morpholine oligomers (PMOs) causing exon-skipping of myostatin increased TA weight and CSA locally in mdx-mice [139]. A follow up study combining systemic treatment with two different PMOs that restored dystrophin and inhibited myostatin, respectively, was promising but the mdx mice receiving the myostatin-inhibiting PMO did not benefit from this treatment alone [119]. A similar study demonstrated similar increases in muscle mass in PMO-skipped myostatin, but also demonstrated that skipping varied among muscles, with the highest level of skipping in the soleus. These studies emphasize the importance of the design of the PMO, as well as the variable results obtained in healthy and mdx animals, suggesting that histopathology plays a role in efficiency of the treatment [119,139].

Recently, it was demonstrated that myostatin knock-out by the means of AAV-Sa-Cas9 gene editing delivered by intramuscular injections increased fiber area and number of fibers per area in aged wild-type mice [79]. However, functional outcomes were not described.

The murine hypermuscular myostatin knockout (Mstn^−/−, also denominated ‘the myostatin-null’) described in 1997, has subsequently been further examined and crossed with various mouse models of neuromuscular diseases. The myostatin-null itself has been described numerous times [1,123,127] with increased muscle and body mass. Force measurements have shown both positive and no effect on absolute force in the myostatin knock-outs but decreased specific force has generally been reported [121,122,123] (Supplementary Table S1). An increased proportion of fast fiber-types has been the common observation [123,124,125,126,127], in line with the findings in studies of pharmacological myostatin inhibition (see above). Another model of myostatin malfunction includes the Compact-mouse (also known as the Berlin High Line BEH^C/C), which contains a 12-bp deletion in the propeptide domain of promyostatin (Mstn^Cmpt-dl1Abc) but leaves the biologically active growth-factor domain of myostatin unaffected [140]. Kocsis and colleagues later found that the Compact genetic background itself, in addition to the promyostatin genetic deletion, determines the phenotype [128] and the use of this model has been rather limited.

A third mouse model is the lean myostatin mouse (Mstn^ln/ln), which has an induced loss-of-function mutation leading to a peptide without the ligand, thus a complete lack of myostatin. This model has similarly increased muscularity but has had most of its use in the field of metabolic research [141].

Crossing myostatin-null with other models of muscular dystrophy has occasionally been the preceding study to pharmacological interventions. Crossing myostatin-null with mdx [129,130] or caveolin-3-deficient mice with transgenic mice overexpressing the myostatin prodomain (“MSTNPro”) [105] did ameliorate the pathological features by increasing body weight, fiber numbers and improving grip strength. However, the crossing of myostatin-null mice with the dy^W/dy^W laminin-deficient mouse model of congenital muscular dystrophy failed to improve the dystrophic phenotype and postnatal lethality was even increased [131].

In addition, a recent study crossing a follistatin overexpressing mouse strain with the calpain 3 knock-out mouse model for LGMD2A led to increased glycolytic muscle mass, but caused the loss of AMP-activated protein kinase signaling, important for contraction-induced glycolysis and poor exercise tolerance [142].