Source: UNIVERSITY OF WYOMING submitted to
MOLECULAR MECHANISMS REGULATING SKELETAL MUSCLE GROWTH AND DIFFERENTIATION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
1009688
Grant No.
(N/A)
Project No.
WYO-574-16
Proposal No.
(N/A)
Multistate No.
NC-_old1184
Program Code
(N/A)
Project Start Date
Apr 21, 2016
Project End Date
Feb 3, 2020
Grant Year
(N/A)
Project Director
Guo, WE.
Recipient Organization
UNIVERSITY OF WYOMING
1000 E UNIVERSITY AVE DEPARTMENT 3434
LARAMIE,WY 82071-2000
Performing Department
Animal Science
Non Technical Summary
A significant proportion of our agricultural crops are devoted to production of food animals and meat products for human consumption. Although much progress has been made in the efficiency of conversion of feedstuffs to muscle growth, the process still needs considerable improvement. Muscle growth is the net effect when protein synthesis exceeds protein degradation. Small changes in rates of either protein synthesisor protein turnover (degradation rate) have profound effects on net protein deposition and rate of growth. In order to increase the efficiency of lean muscle accretion, a better understanding of those basic biological mechanisms in skeletal muscle tissue is necessary. Therefore, we proposed to study multistate research project NC1184 objectives 1. characterize the signal transduction pathways that regulate skeletal muscle growth and metabolism including the influence of endogenous growth factors and various production practicesand 2. characterize the molecular and cellular basis of myogenesis. Previous studies have identified a novel splicing factor, RNA binding motif 20 (RBM20) that is specifically expressed in muscle tissues including cardiac and skeletal muscles. Our published and unpublished data indicate that RBM20 plays a role in muscle differentiation and growth, and RBM20-mediated gene splicing can be regulated by hormones, growth factors and other factors. In our project, we will propose to use a RBM20 deficient rat model to study the cellular and molecular basis of muscle growth and differentiation and the associated signal transduction pathways. The success of the project may provide new insights into muscle growth and develop a novel strategy to improve meat products.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3053999103050%
3053999104050%
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. Characterize the cellular and molecular basis of myogenesis.
Project Methods
Objective 1. Characterize the signal transduction pathways that regulate skeletal muscle growth and metabolism including the influence of endogenous growth factors and various production practices.Previous studies have shown that RBM20 regulates alternative splicing of over 30 genes, among which titin is a major target of RBM20. Our recent publication indicates that in the presence of RBM20, thyroid hormone can regulate titin splicing, however, in the absence of RBM20, hormones cannot regulate titin splicing, suggesting that RBM20 is an essential protein for titin splicing. Interestingly, in the presence of RBM20, titin sizes are different in different muscle types, while in the absence of RBM20, titin express consistent size through all muscle types. Therefore, we hypothesize that endogenous growth factors may influence gene splicing regulation by RBM20 via signaling pathways such as PI3K/Akt/mTOR pathway. Recently, our preliminary data shows that RBM20 is also a major splicing regulator of myosin heavy chain. In cardiac muscle, RBM20 regulates α-myosin to express a larger isoform completely (unpublished data). Therefore, we presume that RBM20 may regulate muscle fiber type switching in skeletal muscle which is important for muscle growth and meat quality. To understand the cellular and molecular mechanisms of RBM20 in the regulation of titin and myosin isoform switching in skeletal muscle, we will use cell cultures and RBM20 deficient rat models treated with growth factors, and examine the isoform switching of these two proteins with special large protein gel and western blot. Next generation sequencing and mass spectrometry will be performed to test the splice variant and transcript profile in different skeletal muscle types, and the posttranslational modifications of RBM20.Objective 2. Characterize the cellular and molecular basis of myogenesis Our previous study has shown that using C2C12 myoblasts for the differentiation of muscle cells, we found that the expression of RBM20 increases from low to medium differentiation and declines in high differentiation which is consistent with sarcomere assembly and maturation. These results suggest that RBM20 is associated with muscle differentiation. Intriguingly, when we treated the RBM20 deficient rats with 60% high fat diet for 5 months, we observed that RBM20 deficient rats did not gain significant weight by comparing to wild type control group, suggesting that RBM20 is also associated with muscle energy metabolism and adipogenesis (unpublished data). Since RBM20 is solely expressed in muscle tissues, we assume that RBM20 may promote energy metabolism and prevent transdifferentiation from muscle to adipose tissues. To test the hypothesis, we will use cell model such as C2C12 and 3T3L1 to differentiate to both muscle cells and adipocytes by knocking down or over expressing RBM20, and test whether RBM20 plays a role in the differentiation of muscle cells and adipocytes. Cell cultures, immunocytochemistry, gene vector construction, cell transfection, fluorescent microscope and transmission electron microscope, Western blot, RT-PCR methods will be applied in this objective.

Progress 04/21/16 to 02/03/20

Outputs
Target Audience: Nothing Reported Changes/Problems:Project director no longer at University of Wyoming. Terminating without final report. What opportunities for training and professional development has the project provided? Nothing Reported 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? n/a

Publications


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

    Outputs
    Target Audience:This project is to gain an in-depth understanding of the molecular regulatory mechanisms in proliferation and differentiation of muscle fibers and muscle growth. Data produced in this project may lead to molecular-based strategies for more efficient production of lean muscle which will benefit both producers and consumers. Research supported by this project will also result in training of new researchers to use molecular and cellular techniques to study muscle proliferation and differentiation in meat-producing animals as well as muscle associated diseases. Dissemination of the results from the research via the timely publication of refereed articles in the peer-reviewed journals will benefit basic research communities. Therefore, the target audiences in this project will be producers, consumers and researchers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provides an opportunity for graduate students to get trained in laboratory research, animal handling, animal surgery and other lab techniques which may assist in their future career development. How have the results been disseminated to communities of interest?The results and findings produced in this project were disseminated by publications in peer-reviewed journals and presentation at scientific meetings as well as in the NC1184 anunal meetings. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we are going to further investigate the role of PI3K/Akt signaling pathway in RBM20-mediated muscle growth and development in different muscle types, and whether RBM20 regulates muscle growth with developmentthrough early muscle differentiation in fetal and/or neonatal stage.

    Impacts
    What was accomplished under these goals? Under these two projects, the Wyoming station is mainly focused on a newly identified splicing factor, RNA binding motif 20 (RBM20) and its role in muscle growth and regeneration. During this reporting period, the accomplishments are listed below: Completed how insulin and T3 affects titin isoform switch in a RBM20-dependent manner in rat skeletal muscle. Investigated how insulin and T3 affects different regions of titin splicing in skeletal muscle and potential molecular signaling pathway behind. Completed titin splicing pattern in different titin band in different type of muscles. Completed muscle injury model in WT and RBM20 KO rats and investigated whether deficiency of RBM20 affects skeletal muscle regeneration. This work has been performed in rat model. Completed how RBM20 deficiency affects skeletal muscle regeneration after injury in rat model. The manuscript is in preparation.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2018 Citation: Chen Z., Maimaiti R., Zhu C., Cai H., Stern A., Mozdziak P., Ge Y., Ford SP., Nathanielsz PW., and Guo W. Z-band and M-band titin splicing and regulation by RBM20 in striated muscles. J Cell Biochem., 2018. doi: 10.1002/jcb.27328. PMID: 30133019.
    • Type: Journal Articles Status: Published Year Published: 2018 Citation: Wei Guo, Chaoqun Zhu, Zhiyong Yin, Qiurong Wang, Mingming Sun, Huojun Cao and Marion L. Greaser. 2018. Splicing factor Rbm20 regulates transcriptional network of titin associated and calcium handling genes in the heart. Int J Biol Sci; 14(4): 369-380. PMID: 29725258.
    • Type: Journal Articles Status: Published Year Published: 2018 Citation: Zhilong Chen, Jiangping Song, Liang Chen, Hanfang Cai, Chaoqun Zhu, Mingming Sun, Allysa Stern, Paul Mozdziak, Ying Ge, Warrie J. Means and Wei Guo. 2018. Characterization of TTN novex splicing variants across species and the role of RBM20 in novex specific exon splicing. Genes (Basel). 9(2): 86-100. PMID: 29438341.
    • Type: Journal Articles Status: Published Year Published: 2018 Citation: Maimaiti Rexiati, Mingming Sun and Wei Guo. 2018. Muscle-specific mis-splicing and heart disease exemplified by RBM20. Genes. 9(1): 18-31. PMID: 29304022.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Wei Guo, Chaoqun Zhu, Vikram Chhatre and Qiurong Wang. Diverse splicing mode of a giant gene TTN and its splicing regulation. The 2nd international Caparica Conference in Splicing, Caparica, Portugal, July 16th-19th 2018.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Chaoqun Zhu, Sreejayan Nair, Jun Ren and Wei Guo. Rbm20 Deficiency Prevents Hypertension and Heart Failure Progression by Regulating Titin Size in Smooth Muscle. 2018 Myofilament Meeting, Madison, WI, May 19th -23rd 2018. Poster# 32.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Mingming Sun, Yutong Jin, Chaoqun Zhu, Maimaiti Rexiati, Hanfang Cai, Zhilong Chen, Martin Liss, Michael Gotthardt, Ying Ge and Wei Guo. Identification of RBM20 Phosphorylation and Its Role in The Regulation of Posttranscriptional Process of Myofilament Gene Titin. 2018 Myofilament Meeting, Madison, WI, May 19th -23rd 2018. Poster# 36.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Wei Guo. Giant elastic protein titin and its splicing regulator RNA binding motif 20 in the onset and development of heart failure. 2018 Myofilament Meeting, Madison, WI, May 19th -23rd 2018. Poster# 55.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Hanfang Cai, Zhilong Chen, Chaoqun Zhu, Rexiati Maimaiti, Qiurong Wang, Vikram Chhatre and Wei Guo. Concerted Splicing Regulation of RBM20 with Splicing Factors in Titin Z-, I- and M-band. 2018 Myofilament Meeting, Madison, WI, May 19th -23rd 2018. Poster# 37.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Maimaiti Rexiati, Mingming Sun and Wei Guo. RBM20 Modulates Myofiber Maturation and Skeletal Muscle Regeneration. 2018 Myofilament Meeting, Madison, WI, May 19th -23rd 2018. Poster# 40.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Rexiati Maimaiti, Zhilong Chen, Chaoqun Zhu, Hanfang Cai, Allysa Stern, Paul Mozdziak, Ying Ge, Stephen P. Ford, Peter W. Nathanielsz and Wei Guo. 2018. Z-band and M-band titin splicing and regulation by RBM20 in striated muscles. The Experimental Biology meeting, San Diego, CA., April 21-25, 2018.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Maimaiti Rexiati, Mingming Sun and Wei Guo. RBM20 deficiency postpones skeletal muscle regeneration after injury and promotes fibrotic tissues formation. The Experimental Biology meeting, San Diego, CA., April 21-25, 2018.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Zhilong Chen and Wei Guo. Splicing variation of TTN novex isoforms across species and RBM20 does not regulate splicing of novex isoforms in cardiac muscle. The Experimental Biology meeting, San Diego, CA., April 21-25, 2018.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Mingming Sun, Yutong Jin, Chaoqun Zhu, Maimaiti Rexiati, Hanfang Cai, ZHilong Chen, Ying Ge and Wei Guo. Mass spectrum analysis of RBM20 phosphorylation and its role in titin splicing. The Experimental Biology meeting, San Diego, CA., April 21-25, 2018.


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

    Outputs
    Target Audience:This project is a basic research project which increases the understanding of the molecular regulatory mechanisms in the regulation of proliferation and differentiation of muscle cells and muscle growth. Data produced in this project may lead to molecular and cellular biology-based strategies for more efficient production of lean meat which will benefit both producers and consumers. Research supported by this project will also result in training of new researchers to use molecular and cellular biological methods to study muscle proliferation and differentiation in meat-producing animals. Dissemination of the results of the research via the timely publication of refereed articles in established journals will benefit basic research communities. Therefore, the target audiences in this project will be producers, consumers and researchers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provides an opportunity for undergraduate students, graduate students and postdocs to get trained in laboratory research, animal handling, animal surgery and other lab techniques which may assist in their future career development. How have the results been disseminated to communities of interest?The results and findings produced in this project were disseminated by publications in peer-reviewed journals and presentation at scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we plan to continue work on the RBM20 knockout model, and characterize the phenotypes caused by RBM20 and determine molecular mechanisms of how RBM20 influences muscle growth and regeneration. We also plan to wrap up and publishtwo papers from the last reporting period.

    Impacts
    What was accomplished under these goals? Accomplishements for Objective 1 RBM20 regulates splicing essential for myofiber structure and skeletal muscle physiology. Completed animal experiments. 3 months and 6 months old WT and RBM20 KO male Sprague-Dawley rats are used to study the differences in myofiber structure and skeletal muscle physiology. Skeletal muscles (longissimus dorsi, soleus, extensor digitorum longus, tibialis anterior and gastrocnemius) were collected. The tissues were snap-frozen in liquid nitrogen and stored at -80 celsius degree or embedded in OCT medium for cryosectioning. Completed the study of RBM20 expression in different types of skeletal mucle (longissimus dorsi, soleus, extensor digitorum longus, tibialis anterior and gastrocnemius). Completed myofiber structural and physiological analysis in skeletal muscle. We studied the change in muscle mass and myofiber cross sectional area between WT and RBM20 KO rat skeletal muscles (soleus, extensor digitorum longus, tibialis anterior). In addition, we studied fibrosis development and sarcolemma integrity in these skeletal muscles. Completed myofilament protein analysis in skeletal muscles. We investigated myosin heavy chain type distribution in WT and RBM20 KO rat skeletal muscles (soleus, extensor digitorum longus, tibialis anterior). Accomplishements for Objective 2 RBM20 mediates skeletal muscle regeneration after injury. Completed animal experiments. 9 weeks old WT and RBM20 KO male Sprague-Dawley rats are used to study skeletal muscle regeneration. Tibialis anterior muscle was injured with the injection of 0.5ml of 1.2% of barium chloride and injured muscles are harvested at 18 h, 3 days, 5days, 7 days, 14 days, 1 month and 2 months post-injury. The other hindlimb tibialis anterior muscle was injected with PBS as control. After harvesting the tissues are liquid nitrogen snap-frozen and stored at -80 celsius degree or embedded in OCT medium for cryosectioning. Completed RBM20 expression during skeletal muscle regeneration. Completed myofiber cross sectional area analysis during skeletal muscle regeneration. Completed analysis of fibrosis level after recovery. Completed analysis of expression of myogenic transcription factors during skeletal muscle regeneration.

    Publications

    • Type: Conference Papers and Presentations Status: Submitted Year Published: 2018 Citation: Mingming Sun, Yutong Jin, Chaoqun Zhu, Maimaiti Rexiati, Hanfang Cai, ZHilong Chen, Ying Ge and Wei Guo. 2017. Mass spectrum analysis of RBM20 phosphorylation and its role in titin splicing. Accepted. The Experimental Biology meeting, San Diego, CA., April 21-25, 2018.
    • Type: Conference Papers and Presentations Status: Submitted Year Published: 2018 Citation: Maimaiti Rexiati, Mingming Sun and Wei Guo. 2017. RBM20 deficiency postpones skeletal muscle regeneration after injury and promotes fibrotic tissues formation. Accepted. The Experimental Biology meeting, San Diego, CA., April 21-25, 2018.
    • Type: Conference Papers and Presentations Status: Submitted Year Published: 2018 Citation: Zhilong Chen and Wei Guo. 2017. Splicing variation of TTN novex isoforms across species and RBM20 does not regulate splicing of novex isoforms in cardiac muscle. Accepted. The Experimental Biology meeting, San Diego, CA., April 21-25, 2018.
    • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Maimaiti Rexiati, Mingming Sun and Wei Guo. 2017. Muscle-specific mis-splicing and heart disease exemplified by RBM20. Genes. Accepted.
    • Type: Journal Articles Status: Published Year Published: 2017 Citation: Guo W, Sun M. 2017. RBM20, a potential target for treatment of cardiomyopathy via titin isoform switching. Biophysical reviews. PMID: 28577155
    • Type: Journal Articles Status: Published Year Published: 2017 Citation: Zhu C, Yin Z, Tan B, Guo W. 2017. Insulin regulates titin pre-mRNA splicing through the PI3K-Akt-mTOR kinase axis in a RBM20-dependent manner. Biochimica et biophysica acta. 1863(9): 2363-2371. PMID: 28676430
    • Type: Journal Articles Status: Published Year Published: 2017 Citation: Zhu C, Guo W. 2017. Detection and quantification of the giant protein titin by SDS-agarose gel electrophoresis. MethodsX. 4:320-327.


    Progress 04/21/16 to 09/30/16

    Outputs
    Target Audience:This project is a basic research project which increases the understanding of the molecular regulatory mechanisms in the regulation of proliferation and differentiation of muscle cells and muscle growth. Data produced in this project may lead to molecular and cellular biology-based strategies for more efficient production of lean meat which will benefit both producers and consumers. Research supported by this project will also result in training of new researchers to use molecular and cellular biological methods to study muscle proliferation and differentiation in meat-producing animals. Dissemination of the results of the research via the timely publication of refereed articles in established journals will benefit basic research communities. Therefore, the target audiences in this project will be producers, consumers and researchers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provides an opportunity for undergraduate students, graduate students and postdocs to get trained on the laboratory research, animal handling, animal sugery and other lab techniques which may assist in their future career development. How have the results been disseminated to communities of interest?The results and findings produced in this project were disseminated by publications in peer-reviewed journals and assist in the goal of improvement of the quality and quantity of meat products for both producers and consumers. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

    Impacts
    What was accomplished under these goals? Under these goals, we have published one paper and one book chapter that are associated with muscle quality and muscle growth. Also we have submitted another article that is related to the signal transduction pathways regulating muscle growth and metabolism. In addition, we produced a rat muscle injury model to study the molecular basis of myogenesis and muscle regeneration.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2016 Citation: Zhu, C., Chen, Z., & Guo, W (2016) Pre-mRNA mis-splicing of sarcomeric genes in heart failure. Biochim Biophys Acta S0925-4439 (16): 30290-3
    • Type: Book Chapters Status: Awaiting Publication Year Published: 2017 Citation: Guo Wei and Greaser, M.L. 2016. Muscle Structure, Proteins and Meat Quality in New aspects of meat quality-from genes to ethics, ed. Peter Purslow, Elsevier, Philadelphia, PA,