Shelley J. Falconer

The decrease in mature myostatin protein in male skeletal muscle is developmentally regulated by growth hormone

Myostatin inhibits myogenesis and there is reduced abundance of the mature protein in skeletal muscles of adult male compared with female mice. This reduction probably occurs after translation, which suggests that it is a regulated mechanism to reduce the availability of myostatin in males. Reduced myostatin may, thereby, contribute to the development of sexually dimorphic growth of skeletal muscle. Our first objective was to determine if the decrease in mature myostatin protein occurs before the linear growth phase to aid growth, or afterwards to maintain the mass of adult muscle. Mice were killed from 2 to 32 weeks and the gastrocnemius muscle was excised. Myostatin mRNA increased from 2 to 32 weeks and was higher in males than females (P < 0.001). In contrast, mature protein decreased in males after 6 weeks (P < 0.001). Our second objective was to determine if growth hormone (GH) induces the decrease in mature myostatin protein. GH increased myostatin mRNA and decreased the abundance of mature protein in hypophysectomised mice (P < 0.05). Our final objective was to determine if the decrease in mature protein occurs in skeletal muscles of male Stat5b−/− mice (Stat5b mediates the actions of GH). As expected, mature myostatin protein was not reduced in Stat5b−/− males compared with females. However, myostatin mRNA remained higher in males than females irrespective of genotype. These data suggest that: (1) the decrease in mature myostatin protein is developmentally regulated, (2) GH acting via Stat5b regulates the abundance of mature myostatin and (3) GH acts via a non‐Stat5b pathway to regulate myostatin mRNA.

Effect of botulinum toxin A-induced paralysis and exercise training on mechanosensing and signalling gene expression in juvenile rat gastrocnemius muscle

Intramuscular injections of the paralytic botulinum neurotoxin A (Btx) and physical exercise are used in the treatment of chronic spasticity in children with cerebral palsy. We tested whether Btx‐induced paralysis and/or exercise training would have differential effects on the expression of mechanosensing and signalling genes implicated in the adaptive remodelling of skeletal muscle. Juvenile (29‐day‐old) male rats were injected with Btx or saline (NoBtx) into the right gastrocnemius and housed in standard cages (NoEx) or with running wheels (Ex), for 3 weeks (n= 6 per group). The mRNA expression of nine sarcomere‐associated genes in the medial gastrocnemius was then determined by quantitative reverse transcriptase‐polymerase chain reaction. The Btx‐injected muscles weighed 50% less than NoBtx muscles, but Ex had no effect on the wet mass of Btx or NoBtx muscles. Atrogenic MuRF1, sarcomeric Titin and myogenic MyoD were upregulated (2‐fold) with the elimination of contractile activity in Btx muscle. Expression of CARP, Ankrd2 and MLP was increased with mechanical stimuli associated with Btx (5‐ to 10‐fold) or Ex (2‐ to 4‐fold). Expression of CARP and Ankrd2 increased synergistically in Btx–Ex muscle (≥20‐fold), indicating that these genes may be sensitive to passive stretch of the sarcomeric I‐band region of titin to which their proteins bind. Tcap, Myopalladin and Atrogin1 were not, or were no longer responsive to the altered mechanical stimuli after 3 weeks of Btx or Ex. The expression of Ankrd2, CARP and MLP may thus be enhanced by passive stretch within the Btx‐paralysed and/or exercising gastrocnemius and contribute to adaptations, other than muscle mass, in juvenile rats.

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.

Translational Signalling, Atrogenic and Myogenic Gene Expression during Unloading and Reloading of Skeletal Muscle in Myostatin-Deficient Mice

Skeletal muscles of myostatin null (Mstn(−/−)) mice are more susceptible to atrophy during hind limb suspension (HS) than are muscles of wild-type mice. Here we sought to elucidate the mechanism for this susceptibility and to determine if Mstn(−/−) mice can regain muscle mass after HS. Male Mstn(−/−) and wild-type mice were subjected to 0, 2 or 7 days of HS or 7 days of HS followed by 1, 3 or 7 days of reloading (n = 6 per group). Mstn(−/−) mice lost more mass from muscles expressing the fast type IIb myofibres during HS and muscle mass was recovered in both genotypes after reloading for 7 days. Concentrations of MAFbx and MuRF1 mRNA, crucial ligases regulating the ubiquitin-proteasome system, but not MUSA1, a BMP-regulated ubiquitin ligase, were increased more in muscles of Mstn(−/−) mice, compared with wild-type mice, during HS and concentrations decreased in both genotypes during reloading. Similarly, concentrations of LC3b, Gabarapl1 and Atg4b, key effectors of the autophagy-lysosomal system, were increased further in muscles of Mstn(−/−) mice, compared with wild-type mice, during HS and decreased in both genotypes during reloading. There was a greater abundance of 4E-BP1 and more bound to eIF4E in muscles of Mstn(−/−) compared with wild-type mice (P<0.001). The ratio of phosphorylated to total eIF2α increased during HS and decreased during reloading, while the opposite pattern was observed for rpS6. Concentrations of myogenic regulatory factors (MyoD, Myf5 and myogenin) mRNA were increased during HS in muscles of Mstn(−/−) mice compared with controls (P<0.001). We attribute the susceptibility of skeletal muscles of Mstn(−/−) mice to atrophy during HS to an up- and downregulation, respectively, of the mechanisms regulating atrophy of myofibres and translation of mRNA. These processes are reversed during reloading to aid a faster rate of recovery of muscle mass in Mstn(−/−) mice.

Undernutrition regulates the expression of a novel splice variant of myostatin and insulin-like growth factor 1 in ovine skeletal muscle.

Undernutrition suppresses the growth of skeletal muscles and alters the expression of insulin-like growth factor 1 (IGF1), a key mitogen, and myostatin, a potent inhibitor of myogenesis. These changes can explain, at least in part, the reduced growth of skeletal muscles in underfed lambs. We have recently identified a myostatin splice variant (MSV) that binds to and antagonizes the canonical signaling of myostatin. In the present study, we hypothesized that the expression of MSV would be reduced in conjunction with myostatin and IGF1 in response to underfeeding in skeletal muscles of sheep. Young growing ewes were fed either ad libitum or an energy-restricted diet (30% of maintenance requirements) for 28 d. This regime of underfeeding resulted in a 24% reduction in body mass (P < 0.001) and a 36% reduction in the mass of the semitendinosus muscles relative to controls (P < 0.001) by day 28. The concentrations of MSV and IGF1 messenger RNA (mRNA) were reduced (both P < 0.001), but myostatin mRNA was not altered in semitendinosus muscles. Unlike the reduced expression of mRNA, the abundance of MSV protein was increased (P < 0.05) and there was no change in the abundance of myostatin protein. Our results suggest that undernutrition for 28 d decreases the signaling of myostatin by increasing the abundance of MSV protein. Although this action may reduce the growth inhibitory activity of myostatin, it cannot prevent the loss of growth of skeletal muscles during undernutrition.

IGF1 stimulates greater muscle hypertrophy in the absence of myostatin in male mice

Insulin-like growth factors (IGFs) and myostatin have opposing roles in regulating the growth and size of skeletal muscle, with IGF1 stimulating, and myostatin inhibiting, growth. However, it remains unclear whether these proteins have mutually dependent, or independent, roles. To clarify this issue, we crossed myostatin null (Mstn-/-) mice with mice overexpressing Igf1 in skeletal muscle (Igf1+) to generate six genotypes of male mice; wild type (Mstn+/+ ), Mstn+/-, Mstn-/-, Mstn+/+:Igf1+, Mstn+/-:Igf1+ and Mstn-/-:Igf1+ Overexpression of Igf1 increased the mass of mixed fibre type muscles (e.g. Quadriceps femoris) by 19% over Mstn+/+ , 33% over Mstn+/- and 49% over Mstn-/- (P < 0.001). By contrast, the mass of the gonadal fat pad was correspondingly reduced with the removal of Mstn and addition of Igf1 Myostatin regulated the number, while IGF1 regulated the size of myofibres, and the deletion of Mstn and Igf1+ independently increased the proportion of fast type IIB myosin heavy chain isoforms in T. anterior (up to 10% each, P < 0.001). The abundance of AKT and rpS6 was increased in muscles of Mstn-/-mice, while phosphorylation of AKTS473 was increased in Igf1+mice (Mstn+/+:Igf1+, Mstn+/-:Igf1+ and Mstn-/-:Igf1+). Our results demonstrate that a greater than additive effect is observed on the growth of skeletal muscle and in the reduction of body fat when myostatin is absent and IGF1 is in excess. Finally, we show that myostatin and IGF1 regulate skeletal muscle size, myofibre type and gonadal fat through distinct mechanisms that involve increasing the total abundance and phosphorylation status of AKT and rpS6.