Figure 1.
Hairpin mediated inhibition of miR-206 results in robust skeletal muscle hypertrophy in vitro
(a) Inhibition of miR-206 in fully differentiated C2C12 myotubes increased cell diameter after 24 and 72 hr of treatment. The fluorescent conjugated control demonstrates the high transfection efficiency. n=4 per group representative of three independent experiments. Scale, 100µm (b) Myotubes were quantified by measuring myotube diameter. Inhibition of miR-206 increased myotube diameter ~25% after 24hr (*, p<0.001 vs. control) and ~90% after 72hr (*, p<0.001 vs. control). n=3 per group representative of three independent experiments (c) Myotube hypertrophy correlated with the dose of miR-206 inhibitor administered. Scale, 100µm (d) miR-206 inhibition increased myotube diameter ~30%, 55% and 95% in response to 40 nM, 100 nM or 200 nM of miR-206 inhibitor (*, all p<0.001 vs. control) n=3 per group representative of three independent experiments. (e) Expression of the hairpin inhibitors resulted in concordant reductions in the transcriptional activity of miR-206 that were significant at 100 nM and 200 nM (*, all p<0.02 vs. control) n=3 per group representative of three independent experiments (f) Ex vivo tyrosine uptake was measured in myotubes treated with miR-206 inhibitor and as compared to cells treated with the negative control (miR-NC), protein synthesis was increased by 20% (*, p=0.003 vs. control) (n=6 per group repeated three times).
Figure 2.
Hypertrophy induced by reduction of miR-206 is regulated by HDAC4.
(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).
Figure 3.
Design and validation of AAV:miRNA-206 and AAV:miR206-sponge vectors.
(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 in vitro 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 in vivo 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).
Figure 4.
The administration of AAV:miR-206 or AAV:miR-206-sponge vectors does not affect post-natal skeletal muscle mass
(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.
Figure 5.
Modulation of miR-206 activity does not regulate skeletal muscle hypertrophy or atrophy
(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.