Discovery of a mammalian splice variant of myostatin that stimulates myogenesis

Myostatin plays a fundamental role in regulating the size of skeletal muscles. To date, only a single myostatin gene and no splice variants have been identified in mammals. Here we describe the splicing of a cryptic intron that removes the coding sequence for the receptor binding moiety of sheep myostatin. The deduced polypeptide sequence of the myostatin splice variant (MSV) contains a 256 amino acid N-terminal domain, which is common to myostatin, and a unique C-terminus of 65 amino acids. Western immunoblotting demonstrated that MSV mRNA is translated into protein, which is present in skeletal muscles. To determine the biological role of MSV, we developed an MSV over-expressing C2C12 myoblast line and showed that it proliferated faster than that of the control line in association with an increased abundance of the CDK2/Cyclin E complex in the nucleus. Recombinant protein made for the novel C-terminus of MSV also stimulated myoblast proliferation and bound to myostatin with high affinity as determined by surface plasmon resonance assay. Therefore, we postulated that MSV functions as a binding protein and antagonist of myostatin. Consistent with our postulate, myostatin protein was co-immunoprecipitated from skeletal muscle extracts with an MSV-specific antibody. MSV over-expression in C2C12 myoblasts blocked myostatin-induced Smad2/3-dependent signaling, thereby confirming that MSV antagonizes the canonical myostatin pathway. Furthermore, MSV over expression increased the abundance of MyoD, Myogenin and MRF4 proteins (P,0.05), which indicates that MSV stimulates myogenesis through the induction of myogenic regulatory factors. To help elucidate a possible role in vivo, we observed that MSV protein was more abundant during early post-natal muscle development, while myostatin remained unchanged, which suggests that MSV may promote the growth of skeletal muscles. We conclude that MSV represents a unique example of intra-genic regulation in which a splice variant directly antagonizes the biological activity of the canonical gene product

Duration of androgen deprivation therapy with postoperative radiotherapy for prostate cancer: a comparison of long-course versus short-course androgen deprivation therapy in the RADICALS-HD randomised trial

Background Previous evidence supports androgen deprivation therapy (ADT) with primary radiotherapy as initial treatment for intermediate-risk and high-risk localised prostate cancer. However, the use and optimal duration of ADT with postoperative radiotherapy after radical prostatectomy remains uncertain. Methods RADICALS-HD was a randomised controlled trial of ADT duration within the RADICALS protocol. Here, we report on the comparison of short-course versus long-course ADT. Key eligibility criteria were indication for radiotherapy after previous radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to add 6 months of ADT (short-course ADT) or 24 months of ADT (long-course ADT) to radiotherapy, using subcutaneous gonadotrophin-releasing hormone analogue (monthly in the short-course ADT group and 3-monthly in the long-course ADT group), daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as metastasis arising from prostate cancer or death from any cause. The comparison had more than 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 75% to 81% (hazard ratio [HR] 0·72). Standard time-to-event analyses were used. Analyses followed intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov , NCT00541047 . Findings Between Jan 30, 2008, and July 7, 2015, 1523 patients (median age 65 years, IQR 60–69) were randomly assigned to receive short-course ADT (n=761) or long-course ADT (n=762) in addition to postoperative radiotherapy at 138 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 8·9 years (7·0–10·0), 313 metastasis-free survival events were reported overall (174 in the short-course ADT group and 139 in the long-course ADT group; HR 0·773 [95% CI 0·612–0·975]; p=0·029). 10-year metastasis-free survival was 71·9% (95% CI 67·6–75·7) in the short-course ADT group and 78·1% (74·2–81·5) in the long-course ADT group. Toxicity of grade 3 or higher was reported for 105 (14%) of 753 participants in the short-course ADT group and 142 (19%) of 757 participants in the long-course ADT group (p=0·025), with no treatment-related deaths. Interpretation Compared with adding 6 months of ADT, adding 24 months of ADT improved metastasis-free survival in people receiving postoperative radiotherapy. For individuals who can accept the additional duration of adverse effects, long-course ADT should be offered with postoperative radiotherapy. Funding Cancer Research UK, UK Research and Innovation (formerly Medical Research Council), and Canadian Cancer Society

Adding 6 months of androgen deprivation therapy to postoperative radiotherapy for prostate cancer: a comparison of short-course versus no androgen deprivation therapy in the RADICALS-HD randomised controlled trial

Background Previous evidence indicates that adjuvant, short-course androgen deprivation therapy (ADT) improves metastasis-free survival when given with primary radiotherapy for intermediate-risk and high-risk localised prostate cancer. However, the value of ADT with postoperative radiotherapy after radical prostatectomy is unclear. Methods RADICALS-HD was an international randomised controlled trial to test the efficacy of ADT used in combination with postoperative radiotherapy for prostate cancer. Key eligibility criteria were indication for radiotherapy after radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to radiotherapy alone (no ADT) or radiotherapy with 6 months of ADT (short-course ADT), using monthly subcutaneous gonadotropin-releasing hormone analogue injections, daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as distant metastasis arising from prostate cancer or death from any cause. Standard survival analysis methods were used, accounting for randomisation stratification factors. The trial had 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 80% to 86% (hazard ratio [HR] 0·67). Analyses followed the intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov, NCT00541047. Findings Between Nov 22, 2007, and June 29, 2015, 1480 patients (median age 66 years [IQR 61–69]) were randomly assigned to receive no ADT (n=737) or short-course ADT (n=743) in addition to postoperative radiotherapy at 121 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 9·0 years (IQR 7·1–10·1), metastasis-free survival events were reported for 268 participants (142 in the no ADT group and 126 in the short-course ADT group; HR 0·886 [95% CI 0·688–1·140], p=0·35). 10-year metastasis-free survival was 79·2% (95% CI 75·4–82·5) in the no ADT group and 80·4% (76·6–83·6) in the short-course ADT group. Toxicity of grade 3 or higher was reported for 121 (17%) of 737 participants in the no ADT group and 100 (14%) of 743 in the short-course ADT group (p=0·15), with no treatment-related deaths. Interpretation Metastatic disease is uncommon following postoperative bed radiotherapy after radical prostatectomy. Adding 6 months of ADT to this radiotherapy did not improve metastasis-free survival compared with no ADT. These findings do not support the use of short-course ADT with postoperative radiotherapy in this patient population

Body mass (mean+sem) for <i>Mstn</i>(−/−) and wild-type mice during seven days of unloading followed by seven days of reloading (n = 6 per genotype and day).

<p>Unlike letters denote significant differences (<i>P</i>&lt;0.05) across days (independent of genotype).</p

Arbitrary concentrations (mean+sem) of MyoD, Myf5 and Myogenin mRNA in the <i>B. femoris</i> muscles of <i>Mstn</i>(−/−) and wild-type mice during seven days of unloading and seven days of reloading (n = 6 per genotype and day).

<p>Asterisks denote differences between genotypes on days shown (*<i>P</i>&lt;0.05, **<i>P</i>&lt;0.01 and ***<i>P</i>&lt;0.001). Unlike letters denote significant differences (<i>P</i>&lt;0.05) across days (independent of genotype).</p

The amount of 4E-BP1 bound to eIF4E.

<p>(A) Representative western blots of 4E-BP1 bound to eIF4E and (B) the ratio (mean+sem) of 4E-BP1 bound to isolated eIF4E purified by an m⁷GTP-sepharose pull-down assay in the <i>gastrocnemius</i> muscle of <i>Mstn</i>(−/−) and wild-type mice (n = 6 per genotype and day) before and after two days of unloading. There were main effects of day (<i>P</i>&lt;0.05) and genotype (<i>P</i>&lt;0.05), but no day × genotype interaction.</p

Changes in the composition of MyHC and the cross-sectional area of muscle fibres.

<p>(A) Representative gels stained with coomassie blue showing the myosin heavy chain (MyHC) protein isoforms in the <i>B. femoris</i> muscle of <i>Mstn</i>(−/−) and wild-type mice during seven days of unloading and seven days of reloading. A mixture of 1∶1 <i>soleus</i> and <i>Extensor digitorum longus</i> served as a ladder. (B) The change (mean+sem) in the relative abundance of type IIb MyHC protein in the <i>B. femoris</i> muscles is shown for <i>Mstn</i>(−/−) and wild-type mice (n = 6 per genotype and day) during seven days of unloading and seven days of reloading. There were main effects of day (<i>P</i>&lt;0.001) and genotype (<i>P</i>&lt;0.01), but no day×genotype interaction. Asterisks denote significant differences between genotypes (*<i>P</i>&lt;0.05, **<i>P</i>&lt;0.01). (C) Cross-sectional area (mean+sem) of myofibres in the <i>gastrocnemius</i> muscle of <i>Mstn</i>(−/−) and wild-type mice during seven days of unloading and seven days of reloading. The cross-sectional area was significantly reduced in both genotypes at d7 (P&lt;0.05), before being restored to pre-unloading areas at d14. Asterisks denote significant differences between genotypes (***<i>P</i>&lt;0.001). Unlike letters denote significant differences (<i>P</i>&lt;0.05) across days (independent of genotype).</p

Arbitrary concentrations (mean+sem) of LC3b, Gabarapl1 and Atg4b mRNA in the <i>B. femoris</i> muscles of <i>Mstn</i>(−/−) and wild-type mice during seven days of unloading and seven days of reloading (n = 6 per genotype and day).

<p>The asterisks denote differences between genotypes on days shown (**<i>P</i>&lt;0.01, ***<i>P</i>&lt;0.01). Unlike letters denote significant differences (<i>P</i>&lt;0.05) across days (independent of genotype).</p

Muscle mass (mean+sem) expressed as a percent of the initial body mass at d0 for <i>Mstn</i>(−/−) and wild-type mice at days 0, 2 and 7 of unloading and days 8, 10 and 14 of reloading.

<p>The asterisks denote differences from d0 within genotype at the days noted (*<i>P</i>&lt;0.05, **<i>P</i>&lt;0.01 and ***<i>P</i>&lt;0.001). The asterisks (**<i>P</i>&lt;0.01) in the data for soleus indicates that muscle mass has been lost equally from both genotypes at d7. EDL = <i>Extensor digitorum longus</i>, Gast = <i>gastrocnemius</i>, Quad = <i>Quadriceps femoris</i>.</p

Alternative splicing of sheep myostatin pre-mRNA and translation of MSV mRNA into protein.

<p>(A) A representative Northern blot identified canonical myostatin (Mstn) and MSV mRNAs in poly(A)⁺ RNA isolated from sheep skeletal muscle using a radiolabeled probe complementary to exon 1 & 2 sequence of sheep myostatin (nt 1–621). (B) Schematic representation of alternative splicing of the myostatin gene. Genomic structure, splicing of canonical myostatin and MSV mRNAs are shown as determined by RT-PCR amplification and sequencing. The sheep myostatin gene has a cryptic third intron sequence (Int 3, 1011 bp) located 21 bp downstream of the intron 2/exon 3 boundary, thereby removing the coding sequence of the canonical mature myostatin protein. Alternate splicing creates a new ORF (966 bp) by appending a novel C-terminal coding sequence (exon 3b, 198 bp) to a truncated propeptide coding sequence of myostatin (exon 1 & 2 and 3a) in the MSV transcript. Open boxes show 5′ and 3′ untranslated regions, filled boxes represent translated sequences. Also shown are exons (Ex), introns (Int), translation start (ATG) and stop (TGA, TAA) sites, and the size of each transcript. Location of the 11 bp deletion in exon 3 identified in Belgian Blue cattle is also indicated. (C) Tissue-specific mRNA expression of MSV and myostatin was analyzed in <i>biceps femoris</i> (Biceps), <i>quadriceps</i> (Quad.) and <i>semitendinosus</i> (Semit.) muscles, and heart, liver, brain, kidney, testicle, ovary, gut, skin and aorta tissues of three months old sheep using RT-PCR. Actin was used as a positive control for each tissue sample. NTC is a no template PCR control. (D) Multiple polypeptide sequence alignment of the predicted C-terminus of MSV in sheep, cattle, pig and dolphin. A consensus proteolytic cleavage site [(K/R)-(X)_n-(K/R)↓ where n = 0, 2, 4, 6 and X is any amino acid except cysteine at aa 271–274] has been identified for precursor convertases. A dotted line indicates the location of the putative cleavage site. The scale shows the positions of the amino acid residues in the MSV polypeptide sequence. The unshaded background highlights residues that are different from the consensus sequence. An <i>in-silico</i> predicted secondary structure of mature sheep MSV is also shown. (E) Schematic representation of the known and proposed proteolytic processing of canonical myostatin and MSV precursors, respectively. The location of the secretion signal peptide and the C-terminal cleavage sites are indicated. Grey filling shows the novel C-terminus of the MSV precursor. Black bars denote the location of polypeptide sequences used to raise MSV-specific polyclonal antibodies (MSVab and MSVab65). (F) Detection of MSV-immunoreactive proteins in <i>semitendinosus</i> muscles of sheep and cattle and its absence in <i>gastrocnemius</i> muscles of mouse and rat (20 µg of total protein per lane) using an anti-MSVab in Western immunoblotting. Recombinant peptide (Recomb.) corresponds to a polypeptide for the C-terminal 65 amino acids (11.9 kDa) of sheep MSV. Molecular weights of a protein marker are also indicated.</p