microRNAs (miRNAs) are short non-coding RNAs that can mediate changes in gene expression and are required for the formation of skeletal muscle (myogenesis). With the goal of identifying novel miRNA biomarkers of muscle disease, we profiled miRNA expression using miRNA-seq in the gastrocnemius muscles of dystrophic mdx4cv mice. After identifying a down-regulation of the miR-30 family (miR-30a-5p, -30b, -30c, -30d and -30e) when compared to C57Bl/6 (WT) mice, we found that overexpression of miR-30 family miRNAs promotes differentiation, while inhibition restricts differentiation of myoblasts in vitro. Additionally, miR-30 family miRNAs are coordinately down-regulated during in vivo models of muscle injury (barium chloride injection) and muscle disuse atrophy (hindlimb suspension). Using bioinformatics tools and in vitro studies, we identified and validated Smarcd2, Snai2 and Tnrc6a as miR-30 family targets. Interestingly, we show that by targeting Tnrc6a, miR-30 family miRNAs negatively regulate the miRNA pathway and modulate both the activity of muscle-specific miR-206 and the levels of protein synthesis. These findings indicate that the miR-30 family may be an interesting biomarker of perturbed muscle homeostasis and muscle disease.

The sarcomeric myosin gene, Myh7b, encodes an intronic microRNA, miR-499, which regulates cardiac and skeletal muscle biology, yet little is known about its transcriptional regulation. To identify the transcription factors involved in regulating Myh7b/miR-499 gene expression, we have mapped the transcriptional start sites and identified an upstream 6.2 kb region of the mouse Myh7b gene whose activity mimics the expression pattern of the endogenous Myh7b gene both in vitro and in vivo. Through promoter deletion analysis, we have mapped a distal E-box element and a proximal Ikaros site that are essential for Myh7b promoter activity in muscle cells. We show that the myogenic regulatory factors, MyoD, Myf5 and Myogenin, bind to the E-box, while a lymphoid transcription factor, Ikaros 4 (Eos), binds to the Ikaros motif. Further, we show that through physical interaction, MyoD and Eos form an active transcriptional complex on the chromatin to regulate the expression of the endogenous Myh7b/miR-499 gene in muscle cells. We also provide the first evidence that Eos can regulate expression of additional myosin genes (Myosin 1 and β-Myosin) via the miR-499/Sox6 pathway. Therefore, our results indicate a novel role for Eos in the regulation of the myofiber gene program.

Striated muscle is a highly specialized collection of tissues with contractile properties that vary according to functional needs. Although muscle fiber types are established postnatally, lifelong plasticity facilitates stimulus-dependent adaptation. Functional adaptation requires molecular adaptation, which is partially provided by miRNA-mediated post-transcriptional regulation. miR-206 is a muscle-specific miRNA enriched in slow muscles. We investigated whether miR-206 drives the slow muscle phenotype or is merely an outcome. We found that miR-206 expression increases in both physiological (including female sex and endurance exercise) and pathological conditions (muscular dystrophy and adrenergic agonism) that promote a slow phenotype. Consistent with that observation, the slow soleus muscle of male miR-206-knockout mice displays a faster phenotype than wild-type mice. Moreover, left ventricles of male miR-206 knockout mice have a faster myosin profile, accompanied by dilation and systolic dysfunction. Thus, miR-206 appears to be necessary to enforce a slow skeletal and cardiac muscle phenotype and to play a key role in muscle sexual dimorphisms.

BackgroundmicroRNA regulation plays an important role in the remodeling that occurs in response to pathologic and physiologic stimuli in skeletal muscle. In response to stress, microRNAs are dynamically regulated, resulting in a widespread “fine-tuning” of gene expression. An understanding of this dynamic regulation is critical to targeting future therapeutic strategies. Experiments elucidating this dynamic regulation have typically relied on in vitro reporter assays, ex vivo sample analysis, and transgenic mouse studies. Surprisingly, no experimental method to date allows rapid in vivo analysis of microRNA activity in mammals.MethodsTo improve microRNA studies we have developed a novel reporter assay for the measurement of skeletal muscle microRNA activity in vivo. To minimize muscle damage, hydrodynamic limb vein injection was used for the introduction of plasmid DNA encoding bioluminescent and fluorescent reporters, including click-beetle luciferase and the far-red fluorescent protein mKATE. We then applied this technique to the measurement of miR-206 activity in dystrophic mdx4cv animals.ResultsWe found that hydrodynamic limb vein injection is minimally damaging to myofibers, and as a result no induction of muscle-specific miR-206 (indicative of an injury response) was detected. Unlike intramuscular injection or electroporation, we found that hydrodynamic limb vein injection results in dispersed reporter expression across multiple hindlimb muscle groups. Additionally, by utilizing click-beetle luciferase from Pyrophorus plagiophthalamus as a reporter and the far-red fluorescent protein mKATE for normalization, we show as a proof of principle that we can detect elevated miR-206 activity in mdx4cv animals when compared to C57Bl/6 controls.ConclusionHydrodynamic limb vein injection of plasmid DNA followed by in vivo bioluminescent imaging is a novel assay for the detection of reporter activity in skeletal muscle in vivo. We believe that this method will allow for the rapid and precise detection of both transcriptional and post-transcriptional regulation of gene expression in response to skeletal muscle stress. Additionally, given the post-mitotic status of myofibers and stable expression of plasmid DNA, we believe this method will reduce biological variability in animal studies by allowing longitudinal studies of the same animal cohort.

11Striated muscle is a highly specialized collection of tissues with contractile properties varying according to 12 functional needs. Although muscle fiber types are established postnatally, lifelong plasticity facilitates stimulus-13 dependent adaptation. Functional adaptation requires molecular adaptation, partially provided by miRNA-14 mediated post-transcriptional regulation. miR-206 is a muscle-specific miRNA enriched in slow muscles. We 15 investigated whether miR-206 drives the slow muscle phenotype or is merely an outcome. We found that miR-16 206 expression increases in both physiologic (including female sex and endurance exercise) and pathologic 17 conditions that promote a slow phenotype. Consistent with that observation, the slow soleus muscle of male miR-18 206 knockout mice displays a faster phenotype than wild-type mice. Moreover, their left ventricles have a faster 19 myosin profile accompanied by male-specific dilation and systolic dysfunction. Thus, miR-206 appears necessary 20 to enforce a slow skeletal and cardiac muscle phenotype and to play a key role in muscle sexual dimorphisms. 21 22 2014). Notably, despite a general sex difference in muscle fiber types, it is not known whether biological sex 72 influences miR-206 expression. Thus, we sought to determine in both sexes whether the miR-206 slow muscle 73 enrichment is an outcome or a driver of the oxidative phenotype. 74 Materials and Methods 75Cloning and mutagenesis: The minimal promoter (minTATA) and MyoG enhancer firefly luciferase reporter gene 76 consructs were previously described (Cheung et al., 2007). We cloned the miR-206 enhancer (GRCm38/mm10 77 chr1: 20,678,678,259) in the same manner as described for MyoG. We generated E-box point mutations 78 (CANNTG à CANNTA) with the QuikChange II site-directed mutagenesis kit (Agilent, 200523, Santa Clara, 79 CA) per the manufacturer's instructions. We verified all clones by Sanger sequencing. MRF expression constructs 80 were a kind gift from Dr. Xuedong Liu (University of Colorado Boulder). Primer sequences are listed in 81 Supplementary Table 1. 82 Cell culture and transfection: We grew C2C12 myoblasts in Growth Medium (GM): high glucose DMEM 83 (Invitrogen, 11960069, Waltham, MA) supplemented with 20% fetal bovine serum, 2 mM L-glutamine, 100 84 U/mL penicillin and 100 μg/mL streptomycin, and 1 mM sodium pyruvate. We differentiated them to myotubes 85 by changing media to Differentiation Medium (DM): high glucose DMEM supplemented with 5% adult horse 86serum, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin, and 1 mM sodium pyruvate. When 87 differentiating, we refreshed DM every day to prevent media acidification. We grew 10T ½ cells in GM but with 88 10% FBS. For luciferase assays, we plated cells in triplicate in 6-well dishes at a density of 50,000 cells/well 89 (C2C12) or 100,000 cells/well (10T ½) 24 hours before transfection. We repeated each experiment at least twice 90 with independent thaws of cells. We transfected with TransIT-LT1 (Mirus Bio, MIR 2305, Madiso...