MYOKINES: AN AVENUE FOR IMPROVED MUSCLE GROWTH

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1024443
• Grant No.: 2021-67016-33718
• Cumulative Award Amt.: $200,000.00
• Proposal No.: 2020-02690
• Project Start Date: Jan 1, 2021
• Project End Date: Dec 31, 2023
• Grant Year: 2021
• Program Code: [A1231]- Animal Health and Production and Animal Products: Improved Nutritional Performance, Growth, and Lactation of Animals

Recipient Organization
UNIV OF IDAHO
875 PERIMETER DRIVE
MOSCOW,ID 83844-9803

Performing Department
(N/A)

Non Technical Summary
Some proteins produced in and released by skeletal muscle regulate growth of the muscle itself and are referred to as; myokines. Myokines have been studied in humans and rodents, but under studied in livestock. Many physiological, genetic and production advancements have improved the efficiency and rate of muscle growth in beef cattle. This proposal investigates a new path towards understanding the regulation of musclegrowth, potentially identifying new natural means of further improving muscle growth in meat animals. This requires evaluation of several myokines at the gene and protein level using harvested bovine muscle stemcells (BSCs). The first step is to characterize the pattern of myokine release from cultured BSCs across normal development and maturation stages. Studies in non-livestock animals indicate that myokines increase following exercise and contribute to beneficial muscle growth associated with contraction. The same mechanisms for induced muscle growthlikely exist in beef cattle and may be promoted in other ways; perhaps even nutritionally. Further, differences in myokine production in muscle cells fromyoung calves versus muscle cells mid-production aged calves, two important times for skeletal muscle growth will be assessed.This will be the first research to provide cattlespecific myokine expression data. Therefore, it will advance in our understanding skeletal muscle growth in beef cattle. This project will provide the insight and data necessary for improvements in muscle growth and efficiency in livestock species using more complete understanding of the natural physiological regulation of muscle growth.We anticipate that this will pave the way for targeted assessment of myokines in live beef cattle, under varied nutritional interventions, within different breeds and species all with the potential to enhance skeletal muscle growth in meat producing livestock.

Animal Health Component

(N/A)

Research Effort Categories

Basic

90%

Applied

(N/A)

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
305	3399	1020	100%

Knowledge Area
305 - Animal Physiological Processes;

Subject Of Investigation
3399 - Beef cattle, general/other;

Field Of Science
1020 - Physiology;

Keywords

muscle

beef

cattle

myokine

satellite cells

Goals / Objectives
Muscle is an endocrine organ and it influences itself in an autocrine/paracrine fashion via synthesis and release of myokines. In studies using mice and rats, exercise appears to be a strong stimulus for myokine release as part of the secretome. Studies in humans and rodents have reported that myokines are important mediators of the physical benefits of exercise and influence muscle hypertrophy and repair. While exercise for production livestock is both impractical and irrelevant, optimal muscle growth may be mediated by myokine production and therefore it is important to evaluate these factors in bovine muscle cells. Moreover, use of β-agonists in finishing beef cattle is known to increase muscle growth and this experiment will examine whether myokine production in bovine muscle cells is influenced by exposure to β-agonists. The AFRI competitive grant program to provide funding for fundamental and applied research this proposal fits within the Animal Nutrition, Growth and Lactation (A1231) program priority. With myokines potentially impacting the extent of muscle growth, these have the potential to improve efficiency and predictability of lean growth, optimization of muscle growth and therefore profitability as muscle becomes the marketable commodity in beef cattle, namely meat. This research will yield data that supports the Multi-State Hatch project (NC1184) Molecular Mechanisms Regulating Skeletal Muscle Growth and Differentiation; where the PD serves the Idaho Research Station and co-PD Dr. Kara Thornton-Kurth represents Utah State. This project will contribute to the understanding of the natural regulatory pathways that control growth of muscle in livestock. The results of this study will support the development of future research projects perhaps even trials that involve manipulation of myokine gene expression using CRISPR/CAS9 or overexpression vectors in vitro in BSCs. Improved efficiency of livestock growth will help feed the global population with high-quality animal protein.Objective 1: Evaluate multiple myokines in primary BSCs harvested from young (under 4 months) and middle age (9-10 month, ~350kg) production beef cattle at the protein and mRNA level.Objective 2: Determine the effect of β-agonists on the temporal expression of myokines relative to untreated cultured primary BSCs. These objectives and aims will test our hypotheses.Aim1: Using standard in vitro culture techniques quantify the temporal (0-96h) expression profiles of myokines produced and released by BSCs as proteins into the culture media as well as the mRNA of myokines in the cultured cells.Aim 2: Compare myokine protein and mRNA abundance and myogenic transcription factors in differentiated and undifferentiated cultured BSCs.Aim 3: Compare myokine temporal expression pattern in cultured untreated BSCs and BSCs treated with physiological doses of an FDA approved β-agonist.

Project Methods
The selected graduate student will make several trips to the lab of Dr. Thornton-Kurth to learn BSC harvest and culture techniques prior to the harvest and collection of these cells at UI in the Fall of 2020. Beef cattle will be sourced from the UI herd and harvested at the UI USDA inspected meats facility.Primary Bovine satellite cells isolation: Bovine satellite cells will be collected from a total of six male, cross-bred beef cattle sourced from the University of Idaho herd. They will be raised similarly, with no prior androgenic or estrogenic implant exposure and will not be direct siblings. Primary BSCs will be harvested from 3 beef calves (under 4 months), and 3 post weaning steers (approx. 9-10 months and ~350 kg.), using the protocol outlined below. This will provide the cell resources required for all experiments proposed in this project. These cells will allow assessment of varied properties and/or attributes of satellite cells from young versus older animals, though importantly, both represent ages during which lean muscle growth is efficient providing nutrition, health and environmental conditions are satisfactory. One animal will be harvested per day allowing time for BSC harvest following administration of captive bolt and exsanguination. BSC extraction is limited by sterile grinder, centrifuge and laminar hood space as well as the importance of timed steps within the protocol. Semimembranosus muscle (~960 g) will be collected and transported to the lab for processing within 30 minutes of harvest. In brief: Isolation of satellite cells will be completed as previously described (Hathaway et al. 1991; Frey et al., 1995). Cells will be stored in liquid nitrogen until use. Clonal analysis of satellite cell cultures established from these preparations demonstrate that between 80 and 90% of the cells were able to establish colonies that contained fusion, indicating that between 80 and 90% of the cells in the preparations are myogenic in nature. The BSC preparations isolated using the procedures described above will be plated on culture dishes precoated with reduced growth factor basement membrane Matrigel® diluted 1:50 with DMEM as recommended.Experiment A: temporal myokine expression in undifferentiated BSCs. Cultures will be plated at a density of approximately 0.2 g/cm2, which has been shown to yield cultures that are approximately 70% confluent after 72 hours in culture (Johnson et al., 1998). All cultures will be plated in DMEM containing 10% FBS and incubated at 37°C, 5% CO2 in a water-saturated environment. After 72 h, cultures will be rinsed once with DMEM and receive fresh media containing 10% FBS; a single lot of FBS will be used to decrease variation that may be present in the TM content of different lots. Immediately prior to collection representative images of the cultured cells will be taken, media will be aspirated and saved for protein extraction, purification and myokine analyses. Because media and Matrigel are by design high in protein content, protein purification will be necessary following total protein extraction from culture media samples. Total RNA extraction from cultured BSCs will be performed for targeted gene expression analyses of myogenic transcription factors (MyoD, Myf5 and myogenin) and myokine specific mRNA abundance. Following 24h of culture on Matrigel, temporal assessment of myokine expression will be assessed at this time 0, and 12, 24, 36, 48, 72 and 96 hours. All experiments will have a minimum of three technical and three experimental replicates for each of the harvested young and mid aged BSCs.Experiment B: temporal myokine expression in differentiated BSCs. As per Experiment A but after 24 hours in culture cells will be induced to differentiate using DMEM with 3% horse serum representing time zero of differentiation. Temporal assessment of myokine expression will be assessed at time 0, 12, 24, 36, 48, 72 and 96 hours. All experiments will have a minimum of three technical and three experimental replicates for each of the harvested young and mid aged BSCs.Experiment C: β-agonist effects on myokine expression. This will entail a temporal study in untreated BSCs versus cultured BSCs treated with one of two doses of β-agonist (10-6 or 10-7 M, Ractopamine-HCl; Cha et al., 2012), comparison of myokine protein levels in the media and myokine mRNA expression as well as mRNA expression of myogenic transcription factors. These will be quantified across various time points between 0, 12, 24, 36, 48, 72 and 96 hours. The BSCs harvested from young calves versus BSCs harvested from mid-aged steers will be evaluated and compared for changes in response magnitude as well as alterations in temporal differences.

Progress 01/01/21 to 12/31/23

Outputs
Target Audience:The research conducted herein was presented at the national annual meetings of the American Society of Animal Sciences in 2022 (poster-2nd place PhD award) and 2023 (oral). Additionally, this work was presented to regional producers at institutional visits and at Extension events. This research was a large component of a PhD thesis that was successfully defended January 2024. The PhD student assigned to this project has two peer-reviewed open-access manuscripts accepted from this work and and two additionalmanuscripts in late stages of preparation for submission. Thereby, the larger scientfic community has been informed of this data. Finally, the PI instructs an upper division and graduate level Endocrinology class, this research was included in the classroom with 75 students registered across 2022 and 2023. Changes/Problems:The first hurdle in this project was associated with a low viable primary muscle cell yield from our first attempts to isolate from our 3-month old harvested steers. This was predominantly due to the small muscle size. We repeated this isloation using additionl young steers and this allowed us to fully execute our research oblectives and aims. One associated note is that due to availability our first harvests were completed at Utah State and the final three steers were harvested at the University of Idaho. The quantification of FNDC5/irisin protein was ineffective, and to date there is not a reliable assay for this for beef species. Our mRNA analyses does confirm that the FNDC5 gene is transcribed in cultured muscle cells. Our final challenge was that our beta-agonist trials showed a modest level of cytotoxicity at the concentrations utilized. Due to the exhaustion of our primary cell supply these experiments were unable to be repeated though they did yield some novel data that will be submitted for publication. What opportunities for training and professional development has the project provided?This research comprised the majority of data associated with the training of a PhD student and preparation of her thesis. This student; Dr. Katie Shira (Walker) successfully defended her PhD-Animal Physiology thesis and is currently a research post-doctoral fellow in a distinguished US academic Molecular genetics lab. She is competent in primary cell harvest, culture, mRNA and protein analyses and is now currently gaining new genetic skills that will facilitate her desire for a career supporting livestock producers using powerful, modern research and applications. Additionally, over the duration of this project three undergraduate students were involved in this project and gained experience in mRNA isolation, protein isolation and real-time PCR analyses. How have the results been disseminated to communities of interest?The research has been published, presented at annual conferences, in the classroom. The peer-reviewed manuscripts have been published in open-access journals with the respective data uploaded and shared. This data has been shared with regional beef producers that are members of regional cattlemens/cattlewomens associations. Finally, the Multistate hatch group NC-1184 have been made aware of this research through the annual meeting and station reports. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1, Aims 1 and 2 were fully realized. We are the first to report the myokine expression profiles in primary cultures of bovine muscle cells harvested from3- month and 11-month old beef steers. This research confirms that myokines are produced and secreted by primary bovine muscle cells in culture. Myokines are important muscle growth regulators that stand to become critical targets for improved growth and efficiency in livestock species. Our researchidentified that both undifferentiated and differentiated BSC express the myokines SPARC, FGF-21, MSTN, DCN, IL-6, IL-15, ERFE, FNDC5, and BDNF. Additionally, BSC also secrete the cognate proteins SPARC, FGF-21, DCN, IL-6, IL-15, ERFE, and BDNF. However, no reliable assays were capable of detecting FNDC5/irisin protein expression. However Objective 2 was executed but resulted in some unanticipated outcomes. More specifically, when the ß-agonist, ractopamine HCl was applied to BSC, expression and secretion of some of these targets were altered, but we observed that when applied at concentrations of 10 µM and 100 µM, ractopamine HCl appears to be toxic to cells and caused increased cell death. Having consumed all of our harvested primary muscle cells we were unable to perform additional experiments with lower doses. This isimportant research to repeat and re-evaluate. Identifying myokines produced in primary culture is a critical first step, and there are many directions this work could progress in the future. This field of study will help link different growth phenotypes to the genetic and physiological mechanisms underpinning these characteristics in livestock species.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: FGF-21 Myokine expression by cultured bovine satellite cells from 3- and 11-month old steers.Oral presentation with one-page published in Journal of Animal Science
• Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Temporal expression of interleukin-6:myokine protein abundance in primary bovine satellite cells from 11-month old beef steers.Poster presentation with abstract published in proceedings.
• Type: Journal Articles Status: Published Year Published: 2024 Citation: Katie A Shira, Brenda M Murdoch, Kara J. Thornton, Caleb C Reicchardt, Gwinyai E. Chibisa, Gordon K Murdoch IL-6, IL-15, CTRP15, and BDNF are myokines produced by cultured bovine satellite cells harvested from Angus steers. Animals 2024, 14(5), 709; https://doi.org/10.3390/ani14050709
• Type: Journal Articles Status: Accepted Year Published: 2024 Citation: Katie A Shira, Kara J. Thornton, Brenda M Murdoch, Gabrielle M Becker, Gwinyai E. Chibisa, Gordon K Murdoch Expression and secretion of SPARC, FGF-21 and DCN in bovine muscle cells: Effects of age and differentiation. Accepted PLoS One February 2024 doi: 10.1371/journal.pone.0299975

Progress 01/01/22 to 12/31/22

Outputs
Target Audience:The preliminary research results have been presented at the Annual Meeting of the American Society of Animal Science. Changes/Problems:Supplies for protein analyses have been delayed due to COVID. What opportunities for training and professional development has the project provided?PhD students Katie Shira (Walker) is appointed to this project and her training is proceeding well. 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?Complete our sample analyses, data analyses and manuscript preparation.

Impacts
What was accomplished under these goals? We have harvested the primary cells from the 3-month and 11-month old beef steers. Primary cell culture experiments have been predominantly completed, with mRNA and protein extractions being completed. Analyses of these samples for myoine expression has started and will be completed soon, in time for data analyses and manuscript preparation

Publications

Progress 01/01/21 to 12/31/21

Outputs
Target Audience:We have established communication with graduate and undergraduate students regrding the objectives and design of this research project. Further, we have presented preliminary data relating to this project amongst the collaborative research team, at graduate seminar and to livestock industry members during recent tours of the laboratory. Changes/Problems:Our initial harvest of satellite cells from our first three young beef calves were modest in yield, slow growing and challenging to culture to confluency. We modified the isolation protocol and harvested additional satellite cells from young cattle and this has yielded much better cells to culture and complete our experiments. We are experienceing some COVId related delays in research supplies including pronase, culture tubes, Elisa's and PCR master mix. We believe that we are managing these challenges effectively. What opportunities for training and professional development has the project provided?One PhD student has been assigned to this project and this research will be the basis of her thesis. We have involved and trained 2 undergraduate students in protein isolation as well as cell culture. Two technicians have learned satellite cell isolation, assessment for contamination and culture methods. Our team has learned that technicall satellite cell isolation can be improved by slight alteration of the protocol and we will be including this in a technical report. We expect to attend the ASAS meeting in Oklahoma, and present some late braking data. How have the results been disseminated to communities of interest?At this stage, data has been disseminated and discussed amongst the research team, colleagues and the graduate students at UI. What do you plan to do during the next reporting period to accomplish the goals?We will be completing additional sattelite cell harvest and isolation, the necessary technical and experimental replicates and the mRNA analyses to fulfill our objectives. The graduate student will be preparing a late-breaking abstract, and comparing the young and middleaged derived satlleite cell responses.

Impacts
What was accomplished under these goals? We have harvested the satellite cells from our young and middle-aged beef cattle. Due to some unforseen challenges with the satellite cell isolation in the young animals we harvested from additional animals. We have utilized in vitro culture to assess the temporal expression patterns of our undifferentiated and differentiated satellite cells and are completing the appropriate number of experimental replicates. We have isoloated protein and are currently isolating mRNA from our samples.We have further initiated our treatments to assess the impacts of B-agonists and obtained some preliminary data. We are currently designing yokine specific primer/probesets for relative quantification of myokine mRNA abundance.

Publications