MOLECULAR MECHANISMS REGULATING SKELETAL MUSCLE GROWTH AND DIFFERENTIATION

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1007294
• Multistate No.: NC-_old1184
• Project Start Date: Oct 1, 2015
• Project End Date: Sep 30, 2020
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF HAWAII
3190 MAILE WAY
HONOLULU,HI 96822

Performing Department
Human Nutrition, Food & Animal Sciences

Non Technical Summary
Improving the efficiency of meat-animal production is critical to sustainable dietary supply of quality proteins to humans with minimal environmental footprints. Evidence indicates that enhancing skeletal muscle growth improves the efficiency of feed utilization, resulting in improved efficiency of meat-animal production. Technologies positively improving skeletal muscle growth process would contribute to enhancement of meat-production efficiency. Recent studies have shown that myostatin (MSTN) is the most potent negative regulator of skeletal muscle growth in animals, suggesting that suppressing MSTN activity will be an ideal strategy to improve skeletal muscle growth. In lab animals, many studies have indeed demonstrated that skeletal muscle growth can be enhanced by inhibiting MSTN activity. In earlier studies, the PI's laboratory has shown that in-ovo suppression of MSTN activity via anti-MSTN antibodies significantly improves post-hatch skeletal muscle growth of broilers, demonstrating that inhibition of MSTN activity is a viable strategy to enhance muscle growth in meat-producing animals. Considering strong consumer sentiment against food products from genetically modified organisms (GMOs), genetic manipulation approaches of MSTN suppression in vivo would be difficult to be adopted as a way of improving muscle growth in meat-producing animals. It was, thus, reasoned that the administration of MSTN inhibitors would be an alternative approach. Therefore, the current project is designed to produce two MSTN-inhibitory proteins (MSTNpro, and follistatin) large enough to be used in animal administration, and to examine the effects of the administration of the proteins on muscle growth in mouse and chicken. Results of the project are expected to provide insights into the potentials of this strategy to improve the production efficiency of meat-producing animals.

Animal Health Component

40%

Research Effort Categories

Basic

30%

Applied

40%

Developmental

30%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
308	3840	1040	25%
305	3220	1040	25%
305	3220	1020	25%
308	3840	1020	25%

Knowledge Area
305 - Animal Physiological Processes; 308 - Improved Animal Products (Before Harvest);

Subject Of Investigation
3220 - Meat-type chicken, live animal; 3840 - Laboratory animals;

Field Of Science
1040 - Molecular biology; 1020 - Physiology;

Keywords

meat animal production

skeletal muscle

myostatin

myostatin inhibitors

mice

chicken

poultry

akt

mtor

Goals / Objectives
Characterize the signal transduction pathways that regulate skeletal muscle growth and metabolism including the influence of endogenous growth factors and various production practices.

Project Methods
Production of Myostatin-inhibitory ProteinsMyostatin-inhibitory proteins, including myostatin propeptide (MSTNPro) and follistatin, will be produced in E. coli. Experiments will be conducted to improve the production yield and potency of these proteins via protein truncation, domain shuffling and sequence modification. The Fc domain of immunoglobulin G will also be fused to the proteins to enhance in vivo biological activities of the proteins. Myostatin-inhibitory activities of the proteins will be examined via reporter gene assay in a cell culture system. Large quantities of selected forms of the proteins, enough for in vivo experiments with mouse and chicken, will be produced.Animal ExperimentAnimal experiments will be carried out to investigate whether the administration of these anti-MSTN proteins enhance skeletal muscle growth of these animals.Mouse experiment: Appropriate number of juvenile mice will be purchased and housed in the UH small animal facility, and be divided into three treatment groups. The three groups will receive weekly, peritoneal injections of recombinant MSTNPro or follistatin at doses of 0, 1 or 10 mg/kg of body weight for 3 months. Body weight and forelimb grip strength will be monitored weekly, and at 1 week after the last injection, animals will be sacrificed, and muscle samples will be collected for histological and biochemical examinations. Histological analysis includes muscle fiber type and size determination and examination of muscle stem cell (satellite cell) proliferation. Expression of genes and protein levels involved in the anabolic Akt/mTOR signaling pathway will also be measured using the real-time PCR and Western blot analysis. The effects of treatment will be analyzed by ANOVA.Chicken experiment: Fertilized broiler eggs (3-day od) will be injected with three different doses (0, 1, 10 ug/egg) of MSTNPro or follistatin in yolk, then post-hatch body weight growth and feed consumption will be monitored for 6 weeks. Following sacrifice, muscle samples will be collected for weight measurement and later biochemical and histochemical analysis. The histological and biochemical assays will be similar to those of mouse experiment. The growout experiment will be performed in chicken growing pen at the UH small animal facility. The effects of treatment will be analyzed by ANOVA.

Progress 10/01/15 to 09/30/20

Outputs
Target Audience:Academics, industrial researchers, and students who are interested in improving the efficiency of meat animal production and meat quality characteristics. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two students were received an MS degree and one student was awarded a Ph.D. degree through the participation of the project. Those students had an opportunity to learn and practice molecular biotechnology skills, protein expression and purification, cell culture techniques, and lab animal handling and sample collection. How have the results been disseminated to communities of interest?Results were published in refereed journals, presented in conferences, and deposited in the form of theses and dissertations. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. To examine the minimum size of myostatin propeptide (MSTNpro) required for full suppression of myostatin activity, various truncated forms of fish myostatin propeptide (fMSTNpro) containing N-terminal maltose-binding protein (MBP) as a fusion partner were expressed in E. coli, and partially purified by affinity chromatography for MSTN-inhibitory activity examination. Results show that the region of flatfish MSTN1pro consisting of residues 45-100 (MBP-fMSTNpro45-100) is sufficient to maintain the full MSTN-inhibitory capacity, indicating that shortened version of MSTN propeptide can be used in various approaches to suppress MSTN activity. Removal of MBP significantly reduced the myostatin-inhibitory potency of fMSTNpro45-100, indicating that MBP as a fusion partner enhances the binding of MBP-fMSTNpro45-100 to myostatin 2. To improve the biopotency of the MBP-fMSTNpro45-100, the Fc domain of mouse immunoglobulin was added to the c-terminal side of MBP-fMSTNpro45-100, and Fc domain-conjugated of MBP-fMSTNpro45-100 (MBP-fMSTNpro45-100mFc) was produced in E. coli and purified. MBP-fMSTNpro45-100mFc showed an equivalent myostatin inhibitory capacity to that of full sequence MSTNpro. 3. A study was designed to examine the effect of neonatal administration MBP-fMSTNpro45-100mFc on skeletal muscle growth in mice. Results showed that either neonatal oral administration or intraperitoneal injection of MSTNpro45-100-Fc did not significantly affect body weight growth and gastrocnemius muscle and organ weights of mice. The results imply that the early neonatal administration of MSTNpro45-100-Fc does not enhance muscle hyperplasia in mice. Another study also showed that the administration of MBP-fMSTNpro45-100mFc for 5 weeks in adult mice did not increase muscle mass. Antibody titer determination showed the presence of antibodies against MBP-fMSTNpro45-100mFc and MBP, suggesting that the antibodies probably neutralized the bioactivity of MBP-fMSTNpro45-100mFc.Interestingly, the injection of MBP-fMSTNpro45-100mFc suppressed Lyme arthritis in a mouse model, in which MSTN expression was highly expressed during arthritis progression, indicating the potential of myostatin inhibition to treat Lyme arthritis. .4. A study was designed to investigate whether myoblast proliferation and differentiation are affected by myostatin inhibition in C2C12 cell culture using MBP-fMSTNpro45-100mFc. The treatment of MBP-MSTNpro45-100-mFc significantly enhanced the proliferation and differentiation of myoblast. The mRNA expression of myogenin, a member of the myogenic regulatory transcription factors (MRFs), was significantly increased by the treatment with MSTNpro45-100mFc in proliferating myoblast culture without affecting other members of MRFs including MyoD, myf5, and MRF4, suggesting that myostatin regulation of myoblast proliferation and differentiation involves myogenin regulation. Furthermore, the examination of signaling molecules associated with myostatin signaling and myogenesis showed that MBP-MSTNpro45-100mFc suppressed myostatin signaling and concurrent changes in signaling involved in myogenesis. Results and indicate that the recombinant MBP-MSTNpro45-100-mFc would be a potentially useful reagent in suppressing myostatin activity in vivo.

Publications

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Academic and industrial researchers and students who are interested in improving the efficiency of meat animal production through myostatin-based biotechnology, as well as who are interested in reducing fat deposition in animal carcasses. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A graduate student had an opportunity to learn and practice molecular biotechnology skills, protein expression and purification, and cell culture techniques. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? A study was designed to investigate whether myoblast proliferation and differentiation is affected by myostatin inhibition in C2C12 cell culture using recombinant myostatin propeptide conjugated to Fc domain of mouse IgG (MSTNpro45-100-mFc) that was produced in the PI's lab. The treatment of MSTNpro45-100-mFc significantly enhanced the proliferation and differentiation of myoblast. The mRNA expression of myogenin, a member of the myogenic regulatory transcription factors (MRFs), was significantly increased by the treatment with MSTNpro45-100-mFc in proliferating myoblast culture without affecting other members of MRFs including MyoD, myf5, and MRF4. Results suggest that myostatin regulation of myoblast proliferation and differentiation involves myogenin regulation and indicate that the recombinant MSTNpro45-100-mFc would be a potentially useful reagent in suppressing myostatin activity in vivo.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Choi, D.H., Yang, J., and Kim, Y.S. 2019. Rapamycin suppresses postnatal muscle hypertrophy induced by myostatin-inhibition accompanied by transcriptional suppression of the Akt/mTOR pathway. BB Reports. 17:182-290.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Academic and industrial researchers and students who are interested in improving the efficiency of meat animal production through myostatin-based biotechnology, as well as who are interested in reducing fat deposition in animal carcasses. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A graduate student had an opportunity to learn and practice molecular biotechnology skills, protein expression and purification, and lab animal handling and samples collection, leading to acquisition of MS degree. How have the results been disseminated to communities of interest?The results were presented in a college-sponsored conference and the NC1184 regional meeting, and deposited in the form of thesis. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? A study was designed to examine the effect of neonatal administration of a truncated form of myostatin propeptide (MSTNpro45-100) conjugated to Fc domain of IgG (MSTNpro45-100-Fc) on skeletal muscle growth in mice. Results showed that either neonatal oral administration or intraperitoneal injection of MSTNpro45-100-Fc did not significantly affect body weight growth and gastrocnemius muscle and organ weights of mice. The results imply that the early neonatal administration of MSTNpro45-100-Fc does not enhance muscle hyperplasia in mice.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Maaeni, Y.M., Lee, S.B., Choi, D.H, Kim, Y.S., and Mozdziak, P. 2018. Cloning of Japanese quail (Coturnix Japonica) follistatin and production of bioactive quail follistatin288 in E. coli. International Journal of Poultry Science. 17(1):8-21.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Academic and industrial researchers and students who are interested in improving the efficiency of meat animal production through myostatin-based biotechnology, as well as who are interested in improving meat quality. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student has an opportunity to learn and practice molecular biotechnology skills. How have the results been disseminated to communities of interest?The results were published in referred journals (Plos One and Journal of Immunology). What do you plan to do during the next reporting period to accomplish the goals?We plan to continue to examine the potentials of the MSTN inhibitor to enhance skeletal muscle growth in selected animal models, as well as to investigate the mechanism(s) of action of the MSTN inhibitor.

Impacts
What was accomplished under these goals? A MSTN-inhibitory molecule, named MBP-fMSTNpro45-100-Fc, was produced and purified by affinity chromatography. Currently, the effect of neonatal administration of the MSTN inhibitor on skeletal muscle growth is examined using mice as an animal model. In a collaborative study, the collaborator at the University of Utah demonstrated that injection of the inhibitor suppressed Lyme arthritis in a mouse model, in which MSTN expression was highly expressed during arthritis progression.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: S.B. Lee, S.K. Park, and Y.S. Kim. 2017. Maltose binding protein-fusion enhances the bioactivity of truncated forms of pig myostatin propeptide produced in E. coli. Plos One 12(4): e0174956.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: J.K. Paquette, Y. Ma, C. Fisher, J. Li, S.B. Lee, J.F. Zachary, Y.S. Kim, C. Teuscher, and J.J. Weis. 2017. Genetic control of Lyme arthritis by Borrelia burgdorferi arthritis-associated locus 1 (Bbaa1) is dependent on localized differential production of IFN-? and requires upregulation of myostatin. The Journal of Immunology. 199(10):3525-3534.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Academic and industrial researchers and students, who are interested in improving the efficiency of meat animal production through myostatin-based biotechnology, as well as who are interested in improving meat quality. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One graduate student and postdoctoral researcher had an opportunity to obtain advanced molecular biology skills through the involvement in the project. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We plan to examine the efficacy of the myostatin-inhibitors in animal models.

Impacts
What was accomplished under these goals? Six different truncated forms of MSTN1pro containing N-terminal maltose binding protein (MBP) as a fusion partner were expressed in E. coli, and partially purified by an affinity chromatography for MSTN-inhibitory activity examination. Peptides covering different regions of flatfish MSTN1pro were also synthesized for MSTN-inhibitory activity examination. Results show that the region of flatfish MSTN1pro consisting of residues 45-100 is sufficient to maintain the full MSTN-inhibitory capacity, implying that shorten version of MSTN propeptide can be used in various approaches to suppress MSTN activity in efforts to improve skeletal muscle growth in meat-producing animals or to ameliorate muscle wasting conditions in humans.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Lee, S.B., Kim, J.H., Jin, D.H., Jin, H.J., and Kim, Y.S. 2016. Myostatin inhibitory region of fish (Paralichthys olivaceus) myostatin-1 propeptide. Comp. Biochem. Physiol. Part B. 194-195:65-70.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Lee, S.B., Park, S.K., and Kim, Y.S. 2015. Production of bioactive chicken (Gallus gallus) follistatin-type proteins in E. coli. AMB Express 5:58. doi 10.1186/s13568-015-0142-3