During embryonic development, progenitor cells arising from the somitic mesoderm commit to a program of differentiation that facilitates the formation of skeletal muscle fibers (termed myogenesis). Under the control of a number of extracellular cues, these myogenic precursors adhere to an orchestrated process of mobilization, proliferation, differentiation, and fusion to create the multi-nucleated myotubes that ultimately become mature fibers (1-3). Many of the most important early changes in gene expression that direct the muscle cell lineage are driven by a family of basic helix-loop-helix transcription factors that includes MyoD, myogenin, MRF4, and Myf5, which are therefore commonly referred to as the muscle regulatory factors (MRFs) 2 (4 -6).TGF-␤ is well characterized as a potent inhibitor of muscle cell differentiation that acts by repressing the transcriptional activity of MRFs (7-9). Interaction between extracellular TGF-␤ and its membrane-bound receptor complex engages a cascade of intracellular signal transduction that promotes nuclear retention of Smad proteins 2 and 3 in complex with Smad4, which subsequently activates or represses hundreds of TGF-␤ target genes (10). Of particular relevance to skeletal muscle cell differentiation, the TGF-␤ signaling protein Smad3 has been shown to physically interact with MRFs in a manner that can inhibit differentiation (9). microRNAs (or miRs) are single-stranded 21-22-nucleotide noncoding RNAs that are capable of controlling gene expression at a post-transcriptional level by stalling the translation of the cognate mRNA or promoting its degradation in a process referred to as RNA interference (RNAi). Here, individual miRs that have been loaded into a specialized collection of interacting proteins referred to as the RNA-induced silencing complex identify and bind to highly specific sequences featuring within exons or the 3Ј-untranslated regions of target mRNAs. The degree of pairing complementarity between a microRNA and its target (as well as target location in the transcript) determine whether translation is subsequently repressed or the transcript is degraded (11,12). In skeletal muscle, specific miRs are increasingly being implicated as key regulators of differentiation, because of their predicted selectivity for genes that are involved in facilitating the myogenic program. Chief among these microRNAs are the so-called "myomiRs," or muscle-enriched microRNAs (including miR-1/206, -133a, and -133b) that are themselves transcribed as targets of MRF activity. As an example, increased miR-206 levels promote myogenic differentiation in vitro (13,14) and in vivo (15, 16), whereas inhibiting miR-206 appears capable of delaying or even preventing myogenic differentiation. Ongoing examination is establishing that additional miRs that are expressed in a variety of cell types including but not exclusive to skeletal muscle may also influence the events of differentiation. For instance, the miR-29 family regulates myogenesis by targeting proteins within the NF-B-YY1 sig...

Smad3/Akt/mTOR/S6K/S6RP signaling plays a critical role in follistatin-mediated muscle growth that operates independently of myostatin-driven mechanisms.

The BMP signaling pathway promotes muscle growth and inhibits muscle wasting via SMAD1/5-dependent signaling.

The Yes-associated protein (YAP) is a core effector of the Hippo pathway, which regulates proliferation and apoptosis in organ development. YAP function has been extensively characterized in epithelial cells and tissues, but its function in adult skeletal muscle remains poorly defined. Here we show that YAP positively regulates basal skeletal muscle mass and protein synthesis. Mechanistically, we show that YAP regulates muscle mass via interaction with TEAD transcription factors. Furthermore, YAP abundance and activity in muscles is increased following injury or degeneration of motor nerves, as a process to mitigate neurogenic muscle atrophy. Our findings highlight an essential role for YAP as a positive regulator of skeletal muscle size. Further investigation of interventions that promote YAP activity in skeletal muscle might aid the development of therapeutics to combat muscle wasting and neuromuscular disorders.

Patients with advanced cancer often succumb to complications arising from striated muscle wasting associated with cachexia. Excessive activation of the type IIB activin receptor (ActRIIB) is considered an important mechanism underlying this wasting, where circulating procachectic factors bind ActRIIB and ultimately lead to the phosphorylation of SMAD2/3. Therapeutics that antagonize the binding of ActRIIB ligands are in clinical development, but concerns exist about achieving efficacy without off-target effects. To protect striated muscle from harmful ActRIIB signaling, and to reduce the risk of off-target effects, we developed an intervention using recombinant adeno-associated viral vectors (rAAV vectors) that increase expression of Smad7 in skeletal and cardiac muscles. SMAD7 acts as an intracellular negative regulator that prevents SMAD2/3 activation and promotes degradation of ActRIIB complexes. In mouse models of cachexia, rAAV:Smad7 prevented wasting of skeletal muscles and the heart independent of tumor burden and serum levels of procachectic ligands. Mechanistically, rAAV:Smad7 administration abolished SMAD2/3 signaling downstream of ActRIIB and inhibited expression of the atrophy-related ubiquitin ligases MuRF1 and MAFbx. These findings identify muscle-directed Smad7 gene delivery as a potential approach for preventing muscle wasting under conditions where excessive ActRIIB signaling occurs, such as cancer cachexia.

microRNAs regulate the development of myogenic progenitors, and the formation of skeletal muscle fibers. However, the role miRNAs play in controlling the growth and adaptation of post-mitotic musculature is less clear. Here, we show that inhibition of the established pro-myogenic regulator miR-206 can promote hypertrophy and increased protein synthesis in post-mitotic cells of the myogenic lineage. We have previously demonstrated that histone deacetylase 4 (HDAC4) is a target of miR-206 in the regulation of myogenic differentiation. We confirmed that inhibition of miR-206 de-repressed HDAC4 accumulation in cultured myotubes. Importantly, inhibition of HDAC4 activity by valproic acid or sodium butyrate prevented hypertrophy of myogenic cells otherwise induced by inhibition of miR-206. To test the significance of miRNA-206 as a regulator of skeletal muscle mass in vivo, we designed recombinant adeno-associated viral vectors (rAAV6 vectors) expressing miR-206, or a miR-206 “sponge,” featuring repeats of a validated miR-206 target sequence. We observed that over-expression or inhibition of miR-206 in the muscles of mice decreased or increased endogenous HDAC4 levels respectively, but did not alter muscle mass or myofiber size. We subsequently manipulated miR-206 levels in muscles undergoing follistatin-induced hypertrophy or denervation-induced atrophy (models of muscle adaptation where endogenous miR-206 expression is altered). Vector-mediated manipulation of miR-206 activity in these models of cell growth and wasting did not alter gain or loss of muscle mass respectively. Our data demonstrate that although the miR-206/HDAC4 axis operates in skeletal muscle, the post-natal expression of miR-206 is not a key regulator of basal skeletal muscle mass or specific modes of muscle growth and wasting. These studies support a context-dependent role of miR-206 in regulating hypertrophy that may be dispensable for maintaining or modifying the adult skeletal muscle phenotype – an important consideration in relation to the development of therapeutics designed to manipulate microRNA activity in musculature.

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Recombinant adeno-associated viral vectors (rAAV vectors) are promising tools for delivering transgenes to skeletal muscle, in order to study the mechanisms that control the muscle phenotype, and to ameliorate diseases that perturb muscle homeostasis. Many studies have employed rAAV vectors carrying reporter genes encoding for β-galactosidase (β-gal), human placental alkaline phosphatase (hPLAP), and green fluorescent protein (GFP) as experimental controls when studying the effects of manipulating other genes. However, it is not clear to what extent these reporter genes can influence signaling and gene expression signatures in skeletal muscle, which may confound the interpretation of results obtained in experimentally manipulated muscles. Herein, we report a strong pro-inflammatory effect of expressing reporter genes in skeletal muscle. Specifically, we show that the administration of rAAV6:hPLAP vectors to the hind limb muscles of mice is associated with dose- and time-dependent macrophage recruitment, and skeletal muscle damage. Dose-dependent expression of hPLAP also led to marked activity of established pro-inflammatory IL-6/Stat3, TNFα, IKKβ and JNK signaling in lysates obtained from homogenized muscles. These effects were independent of promoter type, as expression cassettes featuring hPLAP under the control of constitutive CMV and muscle-specific CK6 promoters both drove cellular responses when matched for vector dose. Importantly, the administration of rAAV6:GFP vectors did not induce muscle damage or inflammation except at the highest doses we examined, and administration of a transgene-null vector (rAAV6:MCS) did not cause damage or inflammation at any of the doses tested, demonstrating that GFP-expressing, or transgene-null vectors may be more suitable as experimental controls. The studies highlight the importance of considering the potential effects of reporter genes when designing experiments that examine gene manipulation in vivo.