Identifying the signalling pathway of a novel Myostatin Splice Variant (MSV)

Myostatin (Mstn), a member of the transforming growth factor-β super family, is a potent negative regulator of skeletal muscle mass. Studies delineating the function of Mstn have identified multiple signal transduction pathways that convey the Mstn signal. Mstn has been shown to influence canonical TGF-β, mitogen activated protein kinase (MAPK) and the PI3K/AKT signal transduction cascades. The discovery in our laboratory of a novel splice variant of Mstn (MSV) that opposes Mstn and stimulates the proliferation of myoblasts provided the impetus for the investigations in this thesis. The splicing of MSV was restricted to the Cetartiodactyl clade of mammals, and MSV may represent an intragenic regulator of Mstn. Thus, the studies undertaken in this thesis were to delineate the signalling pathways used by Mstn and MSV in order to understand how their opposing roles in myoblasts regulate myogenesis. Initially, microarray analysis was used to investigate the transcriptional responses of ovine myoblasts following exposure to recombinant Mstn (eukaryotic) and MSV (prokaryotic) protein. Mstn treatment induced changes in number of transcripts, with changes consistent with previous investigations, for example increased interlukin-6 (IL6) and decreased MyoD. In addition, a novel transcriptional target of Mstn, the β1 subunit of the Na⁺-K⁺-ATPase was discovered. Treatment of ovine myoblasts with recombinant MSV induced a plethora of transcriptional responses. IPA analysis suggested a number of these were due to LPS (endotoxin) contamination, which could be attributed to the production of this protein in E. coli. This was confirmed using the Limulus amebocyte lysate assay. Phase separation using Triton-X 114 proved an effective method for the removal of LPS from the MSV preparation. Western blot analysis was performed following the treatment of ovine myoblasts with Mstn and purified MSV. Consistent with previous myoblast studies Mstn stimulated canonical TGF-β (Smad) signalling and the p38 and ERK components of the MAPK signalling cascade. In contrast to previous studies, Mstn also stimulated AKT signalling, with specific phosphorylation of serine 473 (AKTS⁴⁷³). In addition, Mstn altered the abundance of multiple myogenic transcription factors (MyoD, Myf5, MRF4, Pax7 and Mef2) and the abundance and/or the phosphorylation of targets that have a metabolic role in skeletal muscle (rps6, 4EBP1 and p70S6K). Treatment with MSV increased the abundance of Smad 3, Myf5, 4EBP1 and stimulated AKTS⁴⁷³and 4EBP1 phosphorylation. These data provided the foundation for confirmation of these pathways targeted by MSV in C₂C₁₂ myoblasts that stably expressed MSV. C₂C₁₂ cells expressing MSV had an increased proliferative capacity and showed increased mitochondrial activity (EZ4U assay) as compared to controls. These cells showed an increased abundance of the MRFs MyoD, MRF4 and Myogenin and an increased abundance or phosphorylation of signalling targets involved in canonical TGF-β, MAPK and PI3K/AKT signalling cascades. In addition, cells expressing MSV had an increased abundance and phosphorylation of acetyl co-enzyme A carboxylase (ACC) and 4EBP1, which have established roles in regulating metabolism and the synthesis of protein. The significant overlap of processes influenced by the Na⁺ , K⁺ ATPase complex and Mstn prompted an investigation on how Mstn regulates the β1 subunit of the Na⁺ , K⁺ ATPase. This transcriptional response was found to be dependent on the Smad pathway. In addition, these studies also show that Na⁺ K⁺ ATPase activity plays a role in proliferation and differentiation of ovine myoblasts and suggest that Mstn inhibits ion flow controlled through the function of this enzyme complex. In conclusion, these studies show that Mstn and MSV share a number of common signalling targets. In contrast to previous studies of Mstn, the stable expression MSV increases the activation of AKT signalling and increases the abundance of key myogenic transcription factors. In addition, MSV increases the abundance and phosphorylation of ACC and 4EBP1, molecules involved regulating the synthesis of protein and fatty acids. In addition, the β1 subunit of the Na⁺ K⁺ ATPase, was identified as a novel transcriptional target of Mstn, with this regulation controlled through a Smad dependant mechanism. These data confirm the postulate that Mstn and MSV have divergent signalling functions and suggest a role for MSV in the control of oxidative metabolism

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

Myostatin signals through Pax7 to regulate satellite cell self-renewal

Myostatin, a Transforming Growth Factor-beta (TGF-β) super-family member, has previously been shown to negatively regulate satellite cell activation and self-renewal. However, to date the mechanism behind Myostatin function in satellite cell biology is not known. Here we show that Myostatin signals via a Pax7-dependent mechanism to regulate satellite cell self-renewal. While excess Myostatin inhibited Pax7 expression via ERK1/2 signaling, an increase in Pax7 expression was observed following both genetic inactivation and functional antagonism of Myostatin. As a result, we show that either blocking or inactivating Myostatin enhances the partitioning of the fusion-incompetent self-renewed satellite cell lineage (high Pax7 expression, low MyoD expression) from the pool of actively proliferating myogenic precursor cells. Consistent with this result, over-expression of Pax7 in C2C12 myogenic cells resulted in increased self-renewal through a mechanism which slowed both myogenic proliferation and differentiation. Taken together, these results suggest that increased expression of Pax7 promotes satellite cell self-renewal, and furthermore Myostatin may control the process of satellite cell self-renewal through regulation of Pax7. Thus we speculate that, in addition to the intrinsic factors (such as Pax7), extrinsic factors both positive and negative in nature, will play a major role in determining the stemness of skeletal muscle satellite cells

Proteolytic processing of myostatin is auto-regulated during myogenesis

Myostatin, a potent negative regulator of myogenesis, is proteolytically processed by furin proteases into active mature myostatin before secretion from myoblasts. Here, we show that mature myostatin auto-regulates its processing during myogenesis. In a cell culture model of myogenesis, Northern blot analysis revealed no appreciable change in myostatin mRNA levels between proliferating myoblasts and differentiated myotubes. However, Western blot analysis confirmed a relative reduction in myostatin processing and secretion by differentiated myotubes as compared to proliferating myoblasts. Furthermore, in vivo results demonstrate a lower level of myostatin processing during fetal muscle development when compared to postnatal adult muscle. Consequently, high levels of circulatory mature myostatin were detected in postnatal serum, while fetal circulatory myostatin levels were undetectable. Since Furin proteases are important for proteolytically processing members of the TGF-β superfamily, we therefore investigated the ability of myostatin to control the transcription of furin and auto-regulate the extent of its processing. Transfection experiments indicate that mature myostatin indeed regulates furin protease promoter activity. Based on these results, we propose a mechanism whereby myostatin negatively regulates its proteolytic processing during fetal development, ultimately facilitating the differentiation of myoblasts by controlling both furin protease gene expression and subsequent active concentrations of mature myostatin peptide

Myostatin induces cachexia by activating the ubiquitin proteolytic system through an NF-κB-independent, FoxO1-dependent mechanism

Myostatin, a transforming growth factor-beta (TGF-b) super-family member, has been well characterized as a negative regulator of muscle growth and development. Myostatin has been implicated in several forms of muscle wasting including the severe cachexia observed as a result of conditions such as AIDS and liver cirrhosis. Here we show that Myostatin induces cachexia by a mechanism independent of NF-kB. Myostatin treatment resulted in a reduction in both myotube number and size in vitro, as well as a loss in body mass in vivo. Furthermore, the expression of the myogenic genes myoD and pax3 was reduced, while NF-kB (the p65 subunit) localization and expression remained unchanged. In addition, promoter analysis has confirmed Myostatin inhibition of myoD and pax3. An increase in the expression of genes involved in ubiquitin-mediated proteolysis is observed during many forms of muscle wasting. Hence we analyzed the effect of Myostatin treatment on proteolytic gene expression. The ubiquitin associated genes atrogin-1, MuRF-1, and E214k were upregulated following Myostatin treatment. We analyzed how Myostatin may be signaling to induce cachexia. Myostatin signaling reversed the IGF-1/PI3K/AKT hypertrophy pathway by inhibiting AKT phosphorylation thereby increasing the levels of active FoxO1, allowing for increased expression of atrophy-related genes. Therefore, our results suggest that Myostatin induces cachexia through an NF-kB-independent mechanism. Furthermore, increased Myostatin levels appear to antagonize hypertrophy signaling through regulation of the AKT-FoxO1 pathway

Immunogenic fusion proteins induce neutralizing SARS-CoV-2 antibodies in the serum and milk of sheep

Antigen-specific polyclonal immunoglobulins derived from the serum, colostrum, or milk of immunized ruminant animals have potential as scalable therapeutics for the control of viral diseases including COVID-19. Here we show that the immunization of sheep with fusions of the SARS-CoV-2 receptor binding domain (RBD) to ovine IgG2a Fc domains promotes significantly higher levels of antigen-specific antibodies compared to native RBD or full-length spike antigens. This antibody population contained elevated levels of neutralizing antibodies that suppressed binding between the RBD and hACE2 receptors in vitro. A second immune-stimulating fusion candidate, Granulocyte-macrophage colony-stimulating factor (GM-CSF), induced high neutralizing responses in select animals but narrowly missed achieving significance. We further demonstrated that the antibodies induced by these fusion antigens were transferred into colostrum/milk and possessed cross-neutralizing activity against diverse SARS-CoV-2 variants. Our findings highlight a new pathway for recombinant antigen design in ruminant animals with applications in immune milk production and animal health

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

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

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