Recipient Organization
UNIV OF HAWAII
3190 MAILE WAY
HONOLULU,HI 96822
Performing Department
Human Nutrition, Food & Animal Sciences
Non Technical Summary
In Hawaii, meat animal producers face an increasingly competitive market to satisfy consumer demands for high-quality meat and meat products at an economical price, as well to reduce environmental footprints. Since the feed energy used for fat deposition is wasteful in meat animal production, enhancing skeletal muscle growth and suppressing carcass fat deposition will improve the production efficiency of meat animals. It is, thus, imperative to develop strategies for improving skeletal muscle growth. One strategy of improving skeletal muscle growth is to identify target molecules that are critical in regulating the growth process of skeletal muscles, then modulate the biological activity of the target molecules in a way to maximize the skeletal muscle growth.Recent studies have unequivocally shown that myostatin (MSTN) is the most significant negative regulator of skeletal muscle growth in animals. Unlike other growth factors or hormones regulating animal growth, MSTN demonstrates the exquisite specificity of its action on skeletal muscle, making it an ideal target molecule in modulating skeletal muscle growth. Various MSTN-suppressing molecules have been identified, and these molecules have been shown their potential to improve muscle growth in laboratory animals. However, the potential has not been examined in meat producing animals. To fill this gap, this project is designed (1) To produce and purify large quantities of bioactive MSTN-inhibitory molecules (Fc-fused truncated forms of MSTN-pro proteins), 2) To examine the MSTN-inhibitory capacity of these proteins in in vitro muscle cell culture systems, and 3) To examine the pharmacological characteristics of these proteins in in vivo animal studies. These objectives address the CSREES goal, "enhance economic opportunities for agricultural producers."
Animal Health Component
40%
Research Effort Categories
Basic
40%
Applied
40%
Developmental
20%
Goals / Objectives
The major goal of this project is to enhance skeletal muscle growth of meat-producing animals by modulating the biological activity of myostatin, a potent negative regulator of skeletal muscle growth, through the use of myostatin-inhibitory molecules.
Project Methods
Construction and transformation of expression vectorsThe expression vectors for Fc domain-fused myostatin prodomain (MSTNpro) of pig, chicken, and mouse will be constructed by Gibson cloning following the protocol used in the PI's lab.E. coli strain of K12TB1 (New England Biolab, MA, USA) will be transformed with the expression constructs.Expression and purification of recombinant proteins:Expression-competent E. coli transformed with expression vectors will be grown in 1 L LB media containing ampicillin in 2 L flask at room temperature. When the culture reaches an optical density of 0.3-0.4 Å (600 nm), protein expression will be induced by adding IPTG to a final concentration of 0.4 mM under vigorous shaking. After induction for 6 hours, cell pellet will be collected and lysed, and the soluble fraction will be collected by centrifugation. Soluble fractions will be subject to amylose affinity purification. The amylose affinity purified-fractions of Fc-fused MSTNpro proteins will be further purified by protein G affinity chromatography. The expression and purity will be examined by SDS-PAGE analysis.Myostatin (MSTN)-inhibitory capacity of MSTNpro proteins in vitro systems: HEK293 cells stably expressing (CAGA)12-luciferase gene construct will be used in 96-well culture plate at 40,000 cells/well. Various concentrations (60 - 0 nM) of MBP-MSTNproFc proteins will be examined for their capacity to suppress 1 nM MSTN. The effect of MSTNpro proteins on C2C12 cell proliferation will also be examined. An appropriate concentration of C2C12 myoblasts in DMEM with 10% FBS and 1% antibiotic/antimycotic will be seeded to wells of 96 well plates and grown at 37°C with 5% CO2. At 24 h later, media will be replaced with fresh media containing varying concentrations of the MSTNpro proteins (1,000, 100, 10 and 0 nM) in four replicates of each concentration. Media was replaced every other day with fresh treatment media. Cell proliferation will be examined at 2, 4 and 6 days after the treatment using MTT assay.Plasma clearance of MSTNpro proteins in mouseThe recombinant protein contains MBP and IgG Fc domains as fusion peptides, and the MBP can serve as a useful marker in measuring plasma concentration of the recombinant proteins at various time points (6, 24 and 48 hours) after injection. Using the Western blot assay, the concentration of undigested, intact recombinant protein can be estimated. The density of MSTNpro protein band in the blot will be plotted against time, and the plasma half-life of the recombinant proteins will be estimated.Immune response against the MSTNpro proteins in mouseThe recombinant proteins contain MBP as a fusion partner, and the MBP is a foreign peptide to animals. Thus immune response will be investigated by examining the presence of antibodies against MBP and monitoring animals' health during administration. Recombinant proteins will be administered intraperitoneally to four weaned mice for 4 times with 1 week apart. Blood samples (about 50 uL) will be collected via tail vein at the time of protein administration and 1 week later the last injection by sacrifice. The presence of antibody against the MBP will be examined by ELISA using MBP as a coating antigen.Plasma clearance of MSTNpro proteins in chickenAn intravenous catheter will be placed in the brachial veins of five 4 day-old chicks for protein injection and blood withdrawal. About 100 µg of recombinant protein will be injected, and about 100 µL of blood will be collected 10 min later, then at every 6 hours for 2 days. Plasma proteins will be subjected to SDS-PAGE, and be transferred to a PVDF membrane for immune-blotting with anti-MBP antibodies to quantify the amount of intact recombinant proteins in plasma. The density of the recombinant protein band in the blot will be plotted against time, and the plasma half-life of the recombinant protein will be estimated. The collection at 10 min will serve as the 0 hr recombinant protein level.Administration of recombinant protein to newly-hatched chicksNinety newly-hatched chicks will be purchased from a local hatchery and be equally divided into three groups: no injection, 100 mL sterile phosphate buffer, and 10 mg/kg body wt of recombinant protein dissolved in 100 mL sterile phosphate buffer. The dose is based on a reported study in mice. Chicks with the buffer only and recombinant protein groups will be injected via brachial vein with the solution on day 2, 4, 7, 10 and 14. A pilot study will be conducted before the initiation of this experiment to examine the stressfulness of the injection schedule by measuring the weights of non-injected and saline-injected chicks (20 chicks in each group). The injection schedule may need modification based on the results of the plasma-half-life of recombinant experiment. Chicks will be reared in a standard commercial condition. Weekly body weights and feed consumption will be recorded.Chicks will be sacrificed on day 35 for muscle analysis. Pectoralis, gastrocnemius muscle samples will be isolated immediately after sacrifice, weighed, then about 10 g of muscle samples will be frozen in liquid nitrogen immediately for later analysis of muscle DNA and RNA concentrations and Western blot analysis of molecules involved in signal transduction of MSTN. Patagialis and anterior latissimus dorsi muscles representing fast-twitch and slow-tonic fibers, respectively will be excised, weighed, placed, then a portion of each muscle from the center region of the whole muscle bundle will be taken and frozen in isopentane cooled in liquid nitrogen for later histochemical analysis. Another portion of each muscle will also be frozen in liquid nitrogen for biochemical analysis. Sex will be determined by identification of sexual organs after sacrifice to examine the effect of sex on responsiveness to the administration.Histochemical analysis Cross-sections (16 mm) of patagialis and anterior latissimus dorsi muscles will be cut on a cryostat (-20oC) and mounted onto microscope slides, then stained for myofibrillar adenosine triphosphatase (ATPase) after acid incubation at pH 4.3 and succinic dehydrogenase activity (SDH). Images of stained sections will be captured by a computerized image analysis system available from the PI's lab, and analyzed for Type I (TI), Type IIa (TIIa) and IIb (TIIb) percentage. The ATPase positive fibers will be identified as T1 fibers representing slow-twitch oxidative fibers, and the ATPase negative fibers will be identified as TII. TIIa and TIIb will be separated by inspecting serial sections of ATPase and SDH: TII fibers that are positive to SDH will be identified as TIIa fibers representing fast-twitching oxidative fibers, and TII fibers that are negative to SDH will be identified as TIIb fibers representing fast-twitching glycolytic fibers. The cross-sectional area of each muscle fiber type will be measured using the imaging system. About 500 fibers will be counted to estimate the distribution and size of fiber type.Analysis related to the mechanistic investigationAnterior latissimus dorsi (representing slow-tonic fiber) and one muscle among pectoralis, gastrocnemius and patagialis (representing fast-twitch fiber) that shows the most responsiveness to the recombinant protein will be used for the measurement of DNA and RNA concentrations and expressions of MSTN, myogenic regulatory factors and signaling molecules involved in the Akt/mTOR pathway by real time qPCR and Western blot. The phosphorylation of Akt, 4E-BP1, p70s6K and FoxO will also be measured by Western blot analysis using commercially available antibodies.