PGC-1 alpha and PGC-1 beta increase protein synthesis via ERR alpha in C2C12 myotubes

The transcriptional coactivators peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and PGC-1β are positive regulators of skeletal muscle mass and energy metabolism; however, whether they influence muscle growth and metabolic adaptations via increased protein synthesis is not clear. This study revealed PGC-1α or PGC-1β overexpression in C2C12 myotubes increased protein synthesis and myotube diameter under basal conditions and attenuated the loss in protein synthesis following the treatment with the catabolic agent, dexamethasone. To investigate whether PGC-1α or PGC-1β signal through the Akt/mTOR pathway to increase protein synthesis, treatment with the PI3K and mTOR inhibitors, LY294002 and rapamycin, respectively, was undertaken but found unable to block PGC-1α or PGC-1β’s promotion of protein synthesis. Furthermore, PGC-1α and PGC-1β decreased phosphorylation of Akt and the Akt/mTOR substrate, p70S6K. In contrast to Akt/mTOR inhibition, the suppression of ERRα, a major effector of PGC-1α and PGC-1β activity, attenuated the increase in protein synthesis and myotube diameter in the presence of PGC-1α or PGC-1β overexpression. To characterize further the biological processes occurring, gene set enrichment analysis of genes commonly regulated by both PGC-1α and PGC-1β was performed following a microarray screen. Genes were found enriched in metabolic and mitochondrial oxidative processes, in addition to protein translation and muscle development categories. This suggests concurrent responses involving both increased metabolism and myotube protein synthesis. Finally, based on their known function or unbiased identification through statistical selection, two sets of genes were investigated in a human exercise model of stimulated protein synthesis to characterize further the genes influenced by PGC-1α and PGC-1β during physiological adaptive changes in skeletal muscle

miR-206 Represses Hypertrophy of Myogenic Cells but Not Muscle Fibers via Inhibition of HDAC4

<div><p>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 <i>in vivo</i>, 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.</p> </div

Hypertrophy induced by reduction of miR-206 is regulated by HDAC4.

<p>(a) HDAC4 protein levels were assessed 24hr after transfection of miR-206 inhibitor (hp-miR-206) into differentiated myotubes (*, p=0.0006 vs. control) n=3 per group repeated three times (b) Fully differentiated myotubes were transfected with the NC or hp-miR-206 and after 4 hr treated with vehicle or increasing doses of the HDAC4 inhibitor VPA for 48hr. n=3 per group repeated three times. Myofiber diameter was calculated by measuring the width of at least 200 myotubes per treatment. Scale, 100µm (*, p<0.001 vs. control, ⁺, p<0.001 vs. hp-miR-206). (c) H3 acetylation (*, p<0.05 vs. control) was assessed as a marker of HDAC activity by Western blot (*, p<0.05 vs. control). (d) To confirm the effect of HDAC inhibition by valproic acid (VPA), myotubes were subsequently treated with increasing doses of sodium butyrate (SB) – another HDAC inhibitor - and after 48hr the width of myotubes was assessed (*, p<0.001 vs. control, ⁺, p<0.001 vs. hp-miR-206, ^#, p=0.002 vs. hp-miR-206). Scale, 100µm (e) H3 acetylation levels were assessed via Western blot (*, p<0.05 vs. control).</p

The administration of AAV:miR-206 or AAV:miR-206-sponge vectors does not affect post-natal skeletal muscle mass

<p>(a) HDAC4 protein levels were assessed 28 days after injection of mouse limb muscles with AAV: miR-206 (*, p<0.01 vs. control, n=3-5 per treatment). (b–c) TA muscle mass was assessed 28 days after injection of 1×10⁹ and 1×10¹⁰ vg of AAV: miR-206. No differences were observed in muscle mass or myofiber diameter between treated and control muscles, Myofiber diameter was assessed by measuring the minimum Feret’s diameter. n=4 per treatment. (d) HDAC4 protein levels were examined by Western blot (*, p=0.048 vs. control) in muscles examined 28 days after injection with injected with AAV: miR-206-sponge vector, n=4 per treatment. (e–f) TA muscle mass, and myofiber diameter, were not affected by administration of AAV: miR-206-sponge 28 days. n=4 per treatment.</p

Design and validation of AAV:miRNA-206 and AAV:miR206-sponge vectors.

<p>(a) The design of AAV vectors encoding either miR-206 or a miR-206-sponge (see Methods for details). (b–c) To confirm function, miR-206 and miR-206-sponge constructs were tested for ability to regulate the HDAC4 3’ UTR <i>in vitro</i> using C2C12 cells. After 48 hr, cells were lysed and luciferase activity was measured and normalized to β-gal activity. Whilst AAV: miR-206 inhibited HDAC4 3’ UTR activity by 65% (*, p<0.01 vs. control), the AAV: miR-206-sponge increased the activity of the HDAC4 3’ UTR by 60% (*, p<0.05 vs. control, n=3 per group repeated three times). (d) Administration of 1×10⁹ and 1×10¹⁰ vector genomes of AAV: miR-206 <i>in vivo</i> increased miR-206 transcription approximately 20 fold (*, p=0.002 vs. control) and 100 fold (*, p<0.001 vs. control) respectively, n=3 per group repeated three times. (e) AAV: miR-206 was injected into the TA muscles of mice and after 28 days, miR-133b and miR-1 levels were measured by RT-PCR (p=ND, n=8). (f) AAV: miR-206-sponge vector (1×10¹⁰ vector genomes administered) reduced miR-206 transcripts, as determined by RT-PCR (*, p=0.03 vs. control, n= 4 per group). (g) Whilst the AAV: miR-206-sponge vector (1×10¹⁰ vector genomes administered) reduced miR-206 transcripts (*, p=0.04 vs. control), it did not affect expression of miR-133b or miR-150 (N=ND, n=3 per group).</p

Modulation of miR-206 activity does not regulate skeletal muscle hypertrophy or atrophy

<p>(a) RNA from follistatin expressing muscles undergoing hypertrophy and control muscles was extracted and analyzed via RT-PCR. By 14 days, expression of miRNA-1 (*, p<0.01 vs. control), miR-206 (*, p<0.01 vs. control), and miR-29a (*, p<0.01 vs. control) were decreased, n=6 per treatment. (b) In association with muscle atrophy occurring 7 days after motor nerve resection, miR-206 (*, p<0.01 vs. control) and miR-29a (*, p=0.03 vs. control) expression was increased, whilst miR-133a (*, p=0.02 vs. control) and miR-1 expression was suppressed (*, p=0.02 vs. control). n=6 per treatment (c) Co-administration of AAV: Follistatin-288 and AAV: miR-206 for 28 days did not effect hypertrophy induced by follistatin-288. n=4-7 per treatment (d) Muscle cryosections stained with hematoxylin and eosin demonstrate consistent muscle fiber morphology and no changes in muscle fiber diameter n=8 per treatment. Scale, 100µm (e–f) The TA muscles of mice examined 14 days after nerve resection and administration of either AAV: miR-206 or AAV: miR-206 did not exhibit differences in muscle mass, or myofiber diameter, compared with denervated muscles receiving a control vector. Muscle mass was normalized over initial body weights, n=4 per treatment.</p