Circulating myokine and skeletal muscle transcriptome response to growth and exercise in foals

Hammack, Samantha May

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https://hdl.handle.net/2142/130117 Copy

Description

Title

Circulating myokine and skeletal muscle transcriptome response to growth and exercise in foals

Author(s)

Hammack, Samantha May

Issue Date

2025-06-02

Director of Research (if dissertation) or Advisor (if thesis)

• McCoy, Annette M
• Kersh, Mariana E

Doctoral Committee Chair(s)

McCoy, Annette M

Committee Member(s)

• Dávila, Juanmahel
• Inoue, Makoto

Department of Study

Comparative Biosciences

Discipline

VMS - Comparative Biosciences

Degree Granting Institution

University of Illinois Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• myokine
• equine
• muscle-bone crosstalk
• musculoskeletal
• exercise
• growth
• biomarker

Abstract

Musculoskeletal injuries represent a major threat to equine welfare and are the second leading cause of equine mortality. It is known that exercise, particularly when young, is important for the health of the musculoskeletal system. However, the role of muscle in bone health is largely unknown. Research has shown that the mechanical stimuli exerted on bone by the contraction of muscles leads to corresponding bone adaptation. However, in the last decade, the role of the muscle secretome in crosstalk with bone has been recognized as an important stimulus for bone metabolism. Myokines, proteins produced during muscle activation, appear to be the mechanism by which muscle provides biochemical stimulation of bone. The goal of this study was to further characterize muscle-bone crosstalk via myokines in young horses by 1) measuring systemic levels of myokines known to change in response to exercise in other species and 2) measuring the gene expression of myokines in two opposing muscles of the equine forelimb, the deep digital flexor muscle (DDF) and the common digital extensor muscle (CDE). Given the importance of exercise while young, the focus of this study was foals during the first year of life. Half of the enrolled foals were exercised for 8 weeks starting at 8 weeks of age. Blood was taken every two weeks from 8 weeks of age until the conclusion of exercise and then periodically up until 11 months of age from exercised foals and their age-matched controls. Muscle biopsies and computed tomography (CT) scans were taken at 8 weeks of age (baseline), approximately 5 months of age (4 weeks following the conclusion of exercise), and 8 months of age (approximately 4 months following the conclusion of exercise) in both the exercised and age-matched control foals. Aim 1 of this study was to measure the systemic (blood circulating) protein levels of three myokines, RANK-L, MMP-2, and irisin, known in other species to 1) be related to bone metabolism in some form and 2) change in response to exercise. All three myokines were detected in measurable amounts in the foals. Circulating levels of RANK-L and irisin changed with age. Of the three measured myokines, only irisin was significantly associated with exercise status. This association was only found during the exercise period (8-16 weeks of age) and was lost when data were evaluated over the entire study period, which extended six months beyond the conclusion of the exercise protocol. Unexpectedly, this association between irisin and exercise exhibited a sex-specific response, with exercised females having higher circulating irisin than their male counterparts. This sex-specific irisin exercise response began during the exercise period and continued through the end of the study (11 months of age). The results of this aim suggest that serum irisin measurements hold promise as a minimally invasive biomarker of the musculoskeletal response to exercise and during growth in foals, and RANK-L may be useful as a biomarker for musculoskeletal remodeling during growth. Aim 2 of this study involved measuring myokine gene expression with growth and in response to exercise in foals using bulk RNA sequencing and a best practices transcriptomics bioinformatics pipeline. To control for nuisance variables, the ‘remove unwanted variation’ (RUV) algorithm method was applied. To control for false positive results, a global false discovery rate (FDR) of 0.05 was utilized. Pathway analysis was performed in PANTHER. A literature search yielded a list of 184 candidate myokines that were evaluated independently of global gene expression. Inclusion criteria for this list were 1) previously reported expression in the muscle transcriptome and 2) some suggested mechanistic connection to bone metabolism. The variable responsible for the greatest variation in gene expression across all samples was the type of muscle (DDF or CDE), which accounted for 12% of variance. Differences between the DDF and CDE were apparent when analyzing the data for expression changes with growth as well as in response to exercise. There were also muscle-specific findings in the candidate myokines. The clear differences between muscles at all levels of data analysis highlights the importance of considering which muscle is biopsied when doing muscle gene expression research in the future, and when comparing studies. Skeletal muscle gene expression is not uniform, and differences should be further investigated in order to better understand the role of each muscle in musculoskeletal health. Beyond differences in gene expression between the two muscles, the growth data revealed a shift in gene expression patterns between the two measured time intervals (2-5 months of age and 5-8 months of age) in the study cohort. This correlates with a change in growth rate, previously documented in foals. Foals undergo the fastest change in total mass during the first 6 months of life, after which gains occur at a slower rate. This study is the first to measure longitudinal changes in gene expression of muscles from the same foals over time and represents important baseline data for future muscle gene expression studies in foals. When analyzing changes in candidate myokines in response to exercise, six genes emerged as potential biomarkers for musculoskeletal remodeling in response to exercise: periostin (POSTN), genes for two major collagen 5 components (COL5A1 and COL5A1), insulin-like growth factor 1 (IGF-1), matrix metallopeptidase 14 (MMP-14), and extracellular matrix protein 2 (ECM2). All six myokine genes had expression levels that decreased with growth and increased in response to exercise. All six of these genes encode for myokines that are known to play a role in bone metabolism. This is an important step towards future research to identify reliable biomarkers for response to exercise in foals. An unexpected finding of this work was the sex-specific response to exercise revealed in the levels of circulating irisin (Aim 1), muscle gene expression (Aim 2), and bone properties (seen in CT exams evaluated by collaborators). In the DDF of male foals following the exercise period, there was an increase in gene expression in genes related to cell growth and division, which may indicate a push toward muscle hypertrophy. In the DDF of exercised female foals, there was a modest increase in expression of genes in inflammatory pathways. In the CDE, there was decrease in expression of genes in pathways related to catabolic processes in the exercised male foals, while there was increased expression of these genes in female foals. Among the candidate myokines, POSTN had greater expression in males than females regardless of exercise status. Bone morphogenetic protein 1 (BMP-1) and interleukin 15 (IL-15) also had sex-specific expression within exercised foals. These findings warrant further investigation taking hormone levels into account and will be an important consideration in future research in this field. In conclusion, this research contributed to the field by 1) elucidating differences in gene expression in different muscles of the thoracic limb in foals during growth, in response to exercise, and between sexes, 2) elucidating gene expression in muscles during the period of most rapid growth in foals, 3) elucidating differences in muscle gene expression in foals in response to exercise, 4) elucidating sex-specific changes in muscle gene expression, and 5) identifying 10 potential candidate biomarkers for musculoskeletal changes in foals: irisin, RANK-L, IGF-1, COL5A1, COL5A2, MMP-14, ECM2, POSTN, BMP-1, and IL-15.

Graduation Semester

2025-08

Type of Resource

Thesis

Handle URL

https://hdl.handle.net/2142/130117

Copyright and License Information

Copyright 2025 Samantha May Hammack

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