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

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

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

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

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

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

Surface plasmon resonance kinetic analysis on the binding of MSV to myostatin, ActRIIB to myostatin and myostatin to itself.

$<p>Immobilized onto the flow-cell. Rate constants of association and dissociation interactions (k_a and k_d) and the equilibrium dissociation constants (K_D) of Myostatin, ActRIIB and MSV interactions are shown.</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

Functional analysis of MSV.

<p>(A) Detection of full length MSV mRNA in a stable MSV over-expressing (MSV-line) and an empty vector stably transfected C₂C₁₂ myoblast line (Control-line) using RT-PCR. GAPDH was used as a positive control for each sample. NTC is a no template PCR control. (B) Effect of endogenous over-expression of MSV on the proliferation of C₂C₁₂ myoblasts. Proliferation of the MSV- and Control-line was determined at 0, 24, 48 and 72 h using the WST-1 cell proliferation reagent (**P<0.01, ***P<0.001, n = 8). (C) Effect of rMSV on the proliferation of C₂C₁₂ myoblasts. C₂C₁₂ myoblasts were treated with increasing concentrations of rMSV for 48 h, and cell replication was determined using the WST-1 cell proliferation reagent (*P<0.05, **P<0.01, ***P<0.001, n = 8). (D) Effect of rMSV on the proliferation of sheep myoblasts. Sheep myoblasts were treated with increasing concentrations of rMSV for 48 h, and cell replication was determined using the WST-1 cell proliferation reagent (***P<0.001, n = 8). (E) The abundance of CDK2 protein in nuclear and cytoplasmic fractions of the MSV- and Control-line during proliferation (**P<0.01, n = 3). The abundance of actin and SP-1 proteins was used as cytoplasmic and nuclear loading controls, respectively. (F) The abundance of Cyclin E protein in nuclear and cytoplasmic fractions of the MSV- and Control-line during proliferation (**P<0.01, **P<0.001, n = 3). The abundance of actin and SP-1 proteins was used as cytoplasmic and nuclear loading controls, respectively. (G) The abundance of Myf5, MyoD, Myogenin, MRF4, Pax7 and MEF2 proteins was determined using Western immunoblotting in proliferating myoblasts of the MSV- and Control-line (*P<0.05, n = 3).</p