Biology subjects

Hiam, D.

Publications and source records attributed to Hiam, D..

14 recordsLinked to original sources

bioRxiv2026

Molecular responsiveness to testosterone is uncoupled from skeletal muscle mass and strength in males aged 18 - 80 years

Objective Testosterone exerts wide-ranging physiological effects, including the regulation of anabolic processes in skeletal muscle. However, its role in skeletal muscle ageing remains unclear. This study aimed to establish the association between endogenous testosterone levels and skeletal muscle phenotype across the adult lifespan and determine whether ageing modulates this relationship. Methods Eighty-one healthy males aged 18-80 underwent comprehensive phenotypic assessment. Body composition and muscle morphology were assessed using dual-energy X-ray absorptiometry (DEXA) and peripheral quantitative computed tomography (pQCT). Muscle strength was estimated leg press one-repetition maximum (e1RM). Vastus lateralis biopsies were used to assess fibre type, sequence the transcriptome and establish primary myocytes. Cell lines from young (n=5) and older (n=7) donors were cultured and compared under control and testosterone-treated conditions for cellular phenotype. Results Ageing was associated with lower free testosterone, muscle mass and strength, altered type IIa fibre characteristics, and widespread transcriptional changes in skeletal muscle. Free, but not total, testosterone was strongly associated with the muscle transcriptome, although neither fraction was associated with muscle mass or strength. In primary myocytes, testosterone increased androgen receptor abundance and nuclear localisation and enriched pathways involved in protein translation. Conclusion Testosterone is biologically active in male skeletal muscle, with its free fraction strongly associated with the transcriptome; however, these molecular effects do not translate into differences in muscle mass or strength. Testosterone may therefore play a primary role in establishing the male muscle phenotype during puberty, before transitioning to a more ancillary role in adult skeletal muscle.

physiology↗

bioRxiv2026

Muscle mass and denervation explain variability in maximal power and rapid force across the adult female lifespan

BackgroundDynamic power declines earlier across the lifespan and shows a more pronounced and complex pattern than isometric strength, particularly in ageing females. However, the functional, skeletal muscle and molecular mechanisms underpinning power loss across the female lifespan remain to be collectively examined. MethodsEighty-six females aged 18-80 years and stratified per decade of age completed a series of maximal voluntary knee extensions to construct torque-velocity and power-velocity relationships of the quadriceps. Data points corresponding to >95% maximal power were selected for the evaluation of rate of torque development (RTD) and quadriceps surface electromyography (EMG). Outcomes were quantified within discrete 50ms time bins from torque onset to +200ms and included absolute RTD, RTD normalised to peak force, and EMG amplitude and rate of rise normalised to the maximal compound action potential. Quadriceps morphology was assessed via computed tomography, and a vastus lateralis muscle biopsy was collected to assess markers of denervation and expression of genes associated with the neuromuscular junction and calcium-handling transcriptome. ResultsAgeing led to linear reductions in maximal power (-1.39 {+/-} 0.01% p/year), torque (-0.98 {+/-} 0.13% p/year) and velocity (-0.38 {+/-} 0.01% p/year) (all p < 0.05). Quadriceps skeletal muscle CSA attenuated power loss by [~]40% (p < 0.001), largely through reduction of the decline in torque ([~]50%), with no effect on the decline in velocity. During early time bins, older females generated higher relative RTD accompanied by higher EMG amplitude, whereas during later time bins, older females generated less absolute and relative RTD accompanied by lower EMG amplitude and rate of rise (all p < 0.05). Ageing increased neural cell adhesion molecule (NCAM) positive fibres and fibrosis (both p < 0.05). The presence of NCAM{square} fibres was associated with attenuation of the age-related decline in maximal power ([~]15%), torque ([~]35%) and velocity ([~]60%), suggesting that NCAM{square} fibre prevalence may partially explain the observed age associations. Within the neuromuscular junction transcriptome, ageing reduced acetylcholinesterase and increased laminin alpha-2 and muscle-specific kinase (all FDR < 0.05), whereas lesser changes were observed within the calcium-handling transcriptome. ConclusionsSkeletal muscle CSA explains [~]40% of the age-related decline in quadriceps dynamic maximal power across the female lifespan, whereas a neurodegenerative profile mainly evidenced by age-related changes in voluntary neural drive, denervation and markers of neuromuscular junction instability further contribute to the decline.

physiology↗

bioRxiv2026

MetAR: A semi-automated meta-analysis of skeletal muscle androgen receptors association with age

BackgroundThe maintenance of skeletal muscle health plays a pivotal role in prolonging both the lifespan and healthspan. However, muscle mass and strength exhibit significant declines with age. Ageing is associated with a reduced muscle protein synthesis response to key anabolic stimuli, including the androgen hormone testosterone, termed anabolic resistance. Testosterone enacts its anabolic effects in muscle through androgen receptor (AR) mediated pathways. Emerging evidence suggests that AR availability may represent a rate-limiting factor in androgen signalling, with AR saturation occurring below physiological testosterone levels in some tissues. Prior research in rodents has reported age-related reductions in AR expression, suggesting changes in AR protein content may constitute a key component of anabolic resistance. However, reports of the effects of age on the human skeletal muscle AR are inconclusive and limited by small sample sizes. Therefore, this study aimed to characterise age-related changes in expression of the AR, its regulators and downstream target genes in human skeletal muscle. MethodsWe developed and used a novel R-based pipeline, MetAR, to perform reproducible meta-analyses of publicly available bulk RNA-Seq datasets from NCBI GEO and investigate associations between target gene expression and variables of interest without the need for high-performance computing. Eligible datasets included skeletal muscle samples from healthy adult males aged [≥]18 years, with an age range of [≥] 10 years and sample size [≥] 6. Raw counts data were downloaded, appraised and TMM normalised. Dataset-level associations between age and target gene expression were assessed using linear and generalised additive models (GAMs). Random-effects meta-analyses were performed, and heterogeneity, publication bias and leave-one-out sensitivity assessed. ResultsSixteen skeletal muscle bulk RNA-seq datasets (n = 364; age 18-92 years) were eligible for inclusion in the meta-analyses. AR expression was negatively associated with age ({beta} = -0.006 log2 TMM-CPM per year, p < 0.001) corresponding to a 4.4% decrease in expression per decade. Age was also associated with a significant reduction in expression of various regulators of AR stability, transcriptional activity and nuclear transport. Additionally, steroidogenic enzymes and key downstream targets of the AR, including genes encoding for key structural proteins and mitochondrial function were negatively associated with age. ConclusionsCollectively, these findings suggest a multi-faceted age-associated remodelling of AR expression, signalling and nuclear transport that may contribute to the development of anabolic resistance and consequent age-associated muscle loss.

physiology↗

bioRxiv2026

Tissue-specific divergence in sex-biased DNA methylation across the lifespan

BackgroundAgeing is a sex-specific process characterised by a progressive decline in physiological integrity. DNA methylation represents a primary epigenetic hallmark of ageing, yet sex-specific patterns of epigenetic ageing within and across tissues remain poorly understood. This study aims to address these gaps through an integrated analysis of sex-moderated epigenetic ageing across eight human tissues. MethodsA total of 137 DNA methylation datasets comprising over 36,000 individuals aged 10-114 years were analysed using a meta-analytic workflow to identify age-associated differentially methylated positions (aDMPs) and regions (aDMRs), meta-regression to assess sex moderation, and pathway enrichment analyses to interpret functional relevance. FindingsIndividual tissues displayed distinct age-related methylation trajectories, but some DMP sites showed consistent hyper- or hypomethylation across tissues. Across tissues, we identified 68,630 aDMPs (10%) robustly associated with ageing. Age-associated changes at the regional level were less common, with only 80 robust age-associated aDMRs detected across tissues, representing 0.09% of analysed regions. Sex moderation was observed for only 16 aDMPs (0.002%), indicating that sex effects on age-associated DNA methylation are largely tissue-specific rather than shared across tissues. InterpretationOur findings indicate that age-associated DNA methylation changes predominantly occur at isolated CpG sites rather than extended genomic regions and are strongly dependent on tissue and genomic context. The minimal overlap of sex-moderated methylation signals across tissues suggests that age-related sex differences at the epigenetic level are more likely attributable to tissue- and cell-type-specific variation rather than to broadly conserved epigenetic mechanisms shared across tissues. FundingThis study was funded by an Australian Research Council (ARC) Discovery project (DP200101830). Severine Lamon was funded by an ARC Future Fellowship (FT210100278). Nir Eynon was funded by NHMRC Investigator Grant (APP1194159), and a Hevolution/AFAR New Investigator Award in Aging Biology and Geroscience Research. Mandhri D. Abeysooryia was supported by an Australian Government Research Training Program (RTP) Scholarship. Research in context Evidence before this studyDNA methylation is widely recognised as a central epigenetic hallmark of ageing. Previous research has demonstrated that some age-related methylation changes are conserved across tissues, forming the basis of pan-tissue epigenetic clocks. Most studies to date have primarily examined age effects in isolation. Although biological sex influences ageing trajectories and susceptibility to nearly all age-related diseases, sex-moderated epigenetic ageing has received limited investigation. Specifically, pan-tissue clocks, including GrimAge and PhenoAge, are "sex-aware" but were trained and validated in mixed-sex cohorts, limiting their capacity to disentangle tissue-specific sex effects. Consequently, it remains unclear whether sex-moderated epigenetic ageing signals are shared across tissues or are tissue-specific. Added value of this studyThis study provides a large-scale, comprehensive multi-tissue analysis of sex-moderated epigenetic ageing, integrating 137 DNA methylation datasets across eight human tissues and more than 36,000 male and female individuals spanning the lifespan. Our findings show that while age-associated methylation changes are widespread at the CpG level, sex-moderated effects are rare and largely tissue-specific, with minimal overlap across tissues. Implications of all the available evidenceTogether, the available evidence indicates that epigenetic ageing is predominantly driven by shared, conserved age-related methylation changes, whereas sex differences in epigenetic ageing are modest and context dependent. These sex-related effects are more likely to reflect tissue- and cell-type-specific variation rather than widespread, shared mechanisms. This underscores the need to develop sex-specific epigenetic clocks and to conduct longitudinal cohort and intervention studies to more precisely characterise sex-specific dynamics of epigenetic ageing across tissues.

genetics↗

bioRxiv2024

The transcriptomic signature of age and sex is not conserved in human primary myocytes

BackgroundHuman primary muscle cell (HPMC) lines derived from skeletal muscle biopsies are potentially powerful tools to interrogate the molecular pathways underlying fundamental muscle mechanisms. HPMCs retain their genome in culture, but many endogenous circulating factors are not present in the in vitro environment, or at concentrations that do not mirror physiological levels. To address the assumption that HPMCs are valid models of age and sex-specificity in human muscle research, we examined to what extent HPMC lines retain their source phenotype in culture. MethodsBiopsies from the vastus lateralis muscle were collected from ten males aged 18-30, ten females aged 18-30 and ten males aged 60-75 recruited from a general, healthy population. A portion of the muscle was used for the establishment of 30 individual HMPC lines. The remaining sample was immediately snap frozen and stored for further analysis. RNA was extracted from muscle tissue samples and their corresponding, fully differentiated HMPCs and analysed using RNA Sequencing. To compare their transcriptomic signature, principal component analysis (PCA), differential expression analysis, single-cell deconvolution and pathway enrichment analysis were conducted in R. ResultsA comparison of the transcriptomic signature of 30 human muscle biopsies and their corresponding HPMCs indicated a near-complete lack of retention of the genes and pathways differentially regulated in vivo when compared to their in vitro equivalent, with the exception of several genes encoded on the Y-chromosome. ConclusionsThe diversity of resident cell populations in muscle tissue and the lack of sex- and age-dependent circulating factors in the cellular milieu likely contribute to these observations, which call for caution when using HPMCs as an experimental model of human muscle sex or age.

cell biology↗

bioRxiv2024

The effect of age and sex hormones on female neuromuscular function across the adult lifespan

Neuromuscular ageing is characterized by neural and/or skeletal muscle degeneration that decreases maximal force and power. Female neuromuscular ageing occurs earlier in life compared to males, potentially due to sex hormone changes during the menopausal transition. We quantified neuromuscular function in 88 healthy females represented equally over each decade from 18-80 years of age and investigated the potential role of decreased ovarian hormone concentrations following menopause. Neuromuscular assessment included quadriceps maximal voluntary and evoked isometric torque and surface electromyography measurements, plus one-repetition maximum leg press. Voluntary and evoked torques and one-repetition maximum decreased non-linearly with age, with accelerated reductions starting during the fourth decade. An absence of changes in volitional recruitment of existing quadriceps motor units and Ia afferent facilitation of spinal motoneurons suggests that functional decline was largely mediated by impairment in intrinsic muscle function and/or neuromuscular transmission. Maximal muscle compound action potential amplitude decreased with increasing age for rectus femoris only, indicating increased vulnerability to neuromuscular degeneration compared to vastus lateralis and medialis. In postmenopausal females, some variance in data was explained by inter-individual differences in body composition and physical activity level, however, changes in total or free concentrations of oestrogen, progesterone and/or testosterone were correlated with all age-related decreases in neuromuscular variables. In conclusion, we demonstrate an accelerated onset of neuromuscular degeneration of muscular origin around menopause onset, which is associated with changes in sex hormone concentrations. Interventions aimed at mitigating declines in ovarian hormones and their subsequent effects on neuromuscular function postmenopause should be further explored. Key PointsO_LINeuromuscular deterioration with age is associated with poor physical function and quality of life in older adults, but female-specific trajectories and mechanisms remain unclear. C_LIO_LIThis study is the first to map neuromuscular function across each decade of the adult lifespan in 88 healthy females from 18 to 80 years old and to examine the potential role of hormonal changes after menopause C_LIO_LIWe show an accelerated reduction in neuromuscular function, primarily of muscular origin, that occurs between 40 and 50 years of age, coinciding with the onset of menopause. C_LIO_LIIn postmenopausal females, age-related reductions in neuromuscular function can be explained by differences in body composition, physical activity, and sex hormone concentrations. C_LIO_LIThese findings help us better understand the factors that contribute to the loss of neuromuscular function with age in females, enabling the identification of potential therapeutic interventions to prolong the female health span. C_LI

physiology↗

bioRxiv2024

Mitochondria isolated from male skeletal muscle contain a distinct 1 population of miRNA that are differentially expressed following acute exercise

Initially thought to localise at the cytosol and nucleus only, emerging evidence indicates that miRNAs also localise within the mitochondria where they could regulate diverse pathological and physiological processes. Therefore, the aim of the current study was to use small RNA sequencing to profile and compare the entire population of miRNAs in human skeletal muscle of healthy males in whole-tissue and in isolated mitochondria at rest and in response to acute endurance exercise. Twelve healthy males (age 26 {+/-} 4 years, mean {+/-} SD) cycled for 60 min at 70% VO2peak and muscle biopsies were collected at rest, immediately after and 3 h after exercise. The mitochondria were isolated by immunoprecipitation, further purified, then the resident RNA was sequenced to assess the mitochondrial transcriptome. Small RNA sequencing revealed that mitochondria isolated from male skeletal muscle tissue contain a small and distinct population of miRNAs. Of the approximately 110 mature miRNAs detected in skeletal muscle mitochondria at each time-point, the canonical myo-miRs miR-1, miR-133 and miR-206 families constituted on average 45% of total mitochondria miRNA reads. However, none of these canonical myo-miRs were differentially expressed in mitochondria following endurance exercise. One miRNA, hsa-miR-146b-3p, was differentially expressed in both whole muscle tissue and mitochondria when adjusted for multiple testing (FDR <0.05). Future research is now required to investigate miRNA-mRNA interactions in the mitochondria of skeletal muscle tissue. KEY POINTS SUMMARYO_LIEmerging evidence suggests microRNA are localised in the mitochondria of skeletal muscle cells and may play a role in regulating mitochondrial function. C_LIO_LIWe recently optimised an approach to isolate RNA from mitochondria of human skeletal muscle that is free from contaminating cytosolic RNA and suitable for RNA sequencing. C_LIO_LIIn this study we examined the microRNA population from male skeletal muscle mitochondria before and in the hours following 60 minutes of moderate intensity cycling exercise. C_LIO_LIWe detected around 110 microRNAs in skeletal muscle mitochondria, with the muscle enriched myo-miR such as miR-1, miR-133 and miR-206 families constituting almost half of the reads. However, only one microRNA, hsa-miR-146b-3p, was differentially expressed whereby it increased [~] 10-fold following exercise. C_LIO_LIThe results provide new knowledge into how mitochondria might be regulated at the subcellular level and in response to physiological stressors such as exercise. C_LI

cell biology↗

bioRxiv2024

Total testosterone is not associated with muscle mass, function or exercise adaptations in pre-menopausal females

Key pointsO_LIBioavailable testosterone was positively related to exercise-induced muscle hypertrophy in pre-menopausal females. C_LIO_LINuclear localisation of the androgen receptor was positively related to muscle mass in pre-menopausal females before resistance training, but not with resistance-training induced hypertrophy. C_LIO_LITotal testosterone is not related to muscle mass or strength in pre-menopausal females. C_LIO_LITestosterone treatment induced androgen receptor nuclear translocation, but did not induce mTOR signalling in myocytes from pre-menopausal females. C_LI Testosterone, the major androgen hormone, influences the reproductive and non-reproductive systems in males and females via binding to the androgen receptor (AR). Both circulating endogenous testosterone and muscle AR protein content are positively associated with muscle mass and strength in males, but there is no such evidence in females. Here, we tested whether circulating testosterone levels were associated with muscle mass, function, or the muscle anabolic response to resistance training in pre-menopausal females. Twenty-seven pre-menopausal, untrained females (aged 23.5 {+/-} 4.8) underwent a 12-week resistance training program. Muscle strength, size, power and plasma and urine androgen hormone levels were measured. Skeletal muscle biopsies were collected before and after the training program to quantify the effect of resistance training on AR protein and mRNA content, and nuclear localisation. Primary muscle cell lines were cultured from a subset (n=6) of the participants biopsies and treated with testosterone to investigate its effect on myotube diameter, markers of muscle protein synthesis and AR cellular localisation. Total testosterone was not associated with muscle mass or strength at baseline or with the changes in muscle mass and strength that occurred in response to resistance training. In vitro, supra-physiological doses of testosterone increased myocyte diameter, but this did not occur via the Akt/mTOR pathway as previously suggested. Instead, we show a marked increase in AR nuclear localisation with testosterone administration. In conclusion, we found that, bioavailable testosterone and the proportion of nuclear-localised AR, but not total testosterone, with skeletal muscle mass and strength in pre-menopausal females.

physiology↗

bioRxiv2024

The interactive effect of sustained sleep restriction and resistance exercise on skeletal muscle transcriptomics in young females

IntroductionBoth sleep loss and exercise regulate gene expression in skeletal muscle, yet little is known about how the interaction of these stressors affects the transcriptome. The aim of this study was to investigate the effect of nine nights of sleep restriction, with repeated resistance exercise (REx) sessions, on the skeletal muscle transcriptome of young, trained females. MethodsTen healthy females aged 18-35 years undertook a randomised cross-over study of nine nights sleep restriction (SR; 5-h time in bed) and normal sleep (NS; [≥]7 h time in bed) with a minimum 6-week washout. Participants completed four REx sessions per condition (day 3, 5, 7 and 9). Muscle biopsies were collected both pre- and post-REx on days 3 and 9. Gene and protein expression were assessed by RNA sequencing and Western Blot, respectively. ResultsThree or nine nights of sleep restriction had no effect on the muscle transcriptome independently of exercise. However, close to 3000 transcripts were differentially regulated (FDR < 0.05) 48 h post the completion of three resistance exercise sessions in both NS and SR conditions. Only 39% of downregulated and 18% of upregulated genes were common between both conditions, indicating a moderating effect of sleep restriction on the response to exercise. ConclusionSleep restriction and resistance exercise interacted to alter the enrichment of skeletal muscle transcriptomic pathways in young, resistance-trained females. Performing exercise when sleep restricted may not provide the same adaptive response for individuals as if they were fully rested.

genomics↗

bioRxiv2023

Single-session measures of quadriceps neuromuscular function are reliable in healthy females and unaffected by age

ObjectiveThe inter-session reliability of a wide range of measures used to characterize the aging neuromuscular system is unknown, particularly in females. The aim of this study was to determine the inter-session reliability of quadriceps neuromuscular function assessed via maximal voluntary and evoked force and electromyography responses in healthy young and older females. MethodsTwenty-six females aged 19 - 74 years completed two identical testing sessions 9 {+/-} 7 days apart. Quadriceps neuromuscular function measurements included isometric maximal voluntary force (MVC), high and low frequency twitch force, voluntary and evoked electromyography (EMG) in superficial quadriceps (RMS, M-wave and H-reflex), and maximal torque (T0), velocity (V0) and power (PMAX) derived from torque-velocity and power-velocity relationships. Intra-class correlation coefficients (ICC), coefficients of variation (CoV) and Bland-Altman plots were used to assess inter-session reliability. The effect of participant age on inter-session reliability was assessed by linear regression. ResultsExcellent reliability (ICC > 0.8) was shown for all voluntary and evoked mechanical outcomes and systematic bias was essentially absent. Similarly, all vastus lateralis EMG outcomes showed excellent reliability (ICC > 0.8) with CoVs < 12%, which were better than vastus medialis and rectus femoris outcomes. Participant age was not associated with inter-session reliability (P > 0.05). ConclusionExcellent reliability of voluntary and evoked force and vastus lateralis EMG outcomes measured in healthy females can be attained in one testing session, irrespective of age, increasing feasibility for future research. The random error should however be considered when quantifying age-related differences and/or adaptation to exercise in female neuromuscular function. New and NoteworthyThe test-retest reliability of a diverse range of measures used to quantify neuromuscular function were assessed in younger and older females for the first time. We show that reliable measures of maximal voluntary and evoked quadriceps force and electromyography outcomes can be obtained in one testing session, irrespective of participant age. Thus, neuromuscular function can be accurately assessed across the female lifespan with minimal inconvenience imposed on participants, increasing feasibility for future research.

physiology↗

bioRxiv2023

The influence of Sex on microRNA expression in Human Skeletal Muscle

IntroductionSex differences in microRNA (miRNA) expression profiles have been found across multiple tissues. Skeletal muscle is one of the top tissues that underpin sex-based differences, yet there is limited research into whether there are sex differences in miRNA expression in skeletal muscle. Further, there is limited literature investigating potential differences between males and females in skeletal muscle miRNA expression following exercise, a well-known modulator of miRNA expression. Therefore, the aim of this study was to investigate the effect of sex on miRNA expression in skeletal muscle at baseline and after an acute bout of exercise. MethodsMiRNAs were measured using Taqman(R)miRNA arrays in skeletal muscle of 42 healthy participants from the GeneSMART study (24 males and 20 females aged 18-45 yrs). Differentially expressed miRNAs were identified using mixed linear models adjusted for age. Experimentally validated miRNA gene targets enriched in skeletal muscle were identified in-silico. Over representation analysis was conducted to identify enriched pathways. TransmiR V.2 was used to identify transcription factor (TF)-miR regulatory networks using CHIP-derived data. We further profiled the effects of two sex-biased miRNAs overexpressed in human primary muscle cells lines derived from male and female donors to understand the transcriptome targeted by these miRNAs and investigate and potential sex-specific effects. ResultsA total of 80 miRNAs were differentially expressed in skeletal muscle between the sexes, with 61 miRNAs responding differently to the exercise between the sexes. Sex-biased miRNA gene targets were enriched for muscle-related processes including proliferation and differentiation of the muscle cells and numerous metabolic pathways, suggesting that miRNAs are playing a role in programming sex differences in skeletal muscle. Over-expression of sex-biased miRNAs miRNA-30a and miRNA-30c resulted in profound changes to gene expression profiles that were partly specific to the sex of the cell donor in human primary skeletal muscle cells. ConclusionWe found sex-differences in the expression profile of skeletal muscle miRNAs at baseline and in response to exercise. These miRNAs target regulatory pathways essential to skeletal muscle development and metabolism, suggesting that miRNAs play a profound but highly complex role in programming sex-differences in the skeletal muscle phenotype.

physiology↗

bioRxiv2022

The effect of the menstrual cycle on the circulating microRNA pool in human plasma: a pilot study

Study questionDo ovarian hormones levels influence cf-miRNA expression across the menstrual cycle? Summary answerMeasures of ovarian hormones should be rigorously included in future studies assessing cf-miRNA expression in females and used as time-varying confounders. This exploratory study suggests that cf-miRNAs may play an active role in the regulation of the female cycle in various target tissues. What is known alreadyCell-free or "circulating" miRNAs (cf-miRNAs) are secreted from tissues into most physiological fluids, including plasma, where they play a role in cross-tissue communication. Endogenous and exogenous factors, including sex hormones, regulate cellular miRNA expression levels. Plasma cf-miRNA levels vary with numerous pathological and physiological conditions, including in females. Participants/materials, setting, methodsWe conducted an exploratory study where blood samples were collected from sixteen eumenorrheic females in the early follicular phase, the ovulation phase and the mid-luteal phase of the menstrual cycle. Ovarian hormones oestrogen, progesterone, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured in serum by electrochemiluminescence. The expression levels of 179 plasma-enriched miRNAs were profiled using a PCR-based panel, including stringent internal and external controls to account for the potential differences in RNA extraction and reverse-transcription stemming from low-RNA input samples. Study design, size, durationThis was a prospective monocentric study conducted between March and November 2021. Main results and the role of chanceThis exploratory study suggests that cf-miRNAs may play an active role in the regulation of the female cycle in various target tissues. Linear mixed-models adjusted for the relevant variables showed numerous associations between phases of the menstrual cycle, ovarian hormones and plasma cf-miRNA levels. Validated gene targets of the cf-miRNAs varying with the menstrual cycle were enriched within the female reproductive tissues and primarily involved in cell proliferation and apoptosis. Wider implications of the findingsMeasures of ovarian hormones should be rigorously included in future studies assessing cf-miRNA expression in females and used as time-varying confounders. Limitations, reasons for cautionOur study was conducted on a relatively small cohort of patients. However, it was tightly controlled for endogenous and exogenous confounders, which is critical to ensure robust and reproducible cf-miRNA research. Wider implications of the findingsOur results reinforce the importance of accounting for female-specific biological processes in physiology research by implementing practical or statistical mitigation strategies during data collection and analysis. Study funding/competing interest(s)This study was supported by the clinique romande de readaptation, Sion, Switzerland. Prof. Severine Lamon, is supported by an Australian Research Council Future Fellowship (FT10100278). The authors declare no competing interest Trial registration numberN/A.

physiology↗

bioRxiv2022

DNA methylation and proteomics integration uncover dose-dependent group and individual responses to exercise in human skeletal muscle

ObjectiveExercise is a major regulator of muscle metabolism, and health benefits acquired by exercise are a result of molecular shifts occurring across multiple OMIC levels (i.e. epigenome, transcriptome, proteome). Identifying robust targets associated with exercise response, at both group and individual levels, is therefore important to develop health guidelines and targeted health interventions. MethodsTwenty, apparently healthy, moderately trained (VO2 max= 51.0{+/-}10.6 mL{middle dot}min-1{middle dot}kg-1) males (age range= 18-45yrs) from the Gene SMART (Skeletal Muscle Adaptive Responses to Training) study completed a 12-week High-Intensity Interval Training (HIIT) intervention. Muscle biopsies were collected at baseline and after 4, 8, and 12 weeks of HIIT. High throughput DNA methylation ([~]850 CpG sites), and proteomic ([~]3000 proteins) analyses were conducted at all-time points. Mixed-models were applied to estimate group and individual changes, and methylome and proteome integration was conducted using a holistic multilevel approach with the mixOmics package. ResultsSignificant shifts in the methylome (residual analysis) and proteome profiles were observed after 12 weeks of HIIT. 461 proteins significantly changed over time (at 4, 8, and 12 weeks), whilst only one differentially methylated position (DMP) was changed (adj.p-value <0.05). K-means analysis revealed clear protein clustering exhibiting similar changes over time. Individual responses to training were observed in 101 proteins. Seven proteins had a large effect-sizes >0.5, among them are two novel exercise-related proteins, LYRM7 and EPN1. Integration analysis uncovered bidirectional relationships between the methylome and proteome. ConclusionsWe showed a significant influence of HIIT on the epigenome and proteome in human muscle, and uncovered groups of proteins clustering according to similar patterns across the exercise intervention. Individual responses to exercise were observed in the proteome with novel mitochondrial and metabolic proteins consistently changed across individuals. Future work is required to elucidate the role of such proteins in response to exercise as well as to investigate the mechanisms associating genes and proteins in response to exercise.

molecular biology↗

bioRxiv2021

Genome-wide DNA methylation and transcriptome integration reveal distinct sex differences in skeletal muscle.

Nearly all human complex traits and diseases exhibit some degree of sex differences, with epigenetics being one of the main contributing factors. Various tissues display sex differences in DNA methylation, however this has not yet been explored in skeletal muscle, despite skeletal muscle being among the tissues with the most transcriptomic sex differences. For the first time, we investigated the effect of sex on autosomal DNA methylation in human skeletal muscle across three independent cohorts (Gene SMART, FUSION, and GSE38291) using a meta-analysis approach, totalling 369 human muscle samples (222 males, 147 females), and integrated this with known sex-biased transcriptomics. We found 10,240 differentially methylated regions (DMRs) at FDR < 0.005, 94% of which were hypomethylated in males, and gene set enrichment analysis revealed that differentially methylated genes were involved in muscle contraction and substrate metabolism. We then investigated biological factors underlying DNA methylation sex differences and found that circulating hormones were not associated with differential methylation at sex-biased DNA methylation loci, however these sex-specific loci were enriched for binding sites of hormone-related transcription factors (with top TFs including androgen (AR), estrogen (ESR1), and glucocorticoid (NR3C1) receptors). Fibre type proportions were associated with differential methylation across the genome, as well as across 16 % of sex-biased DNA methylation loci (FDR < 0.005). Integration of DNA methylomic results with transcriptomic data from the GTEx database and the FUSION cohort revealed 326 autosomal genes that display sex differences at both the epigenome and transcriptome levels. Importantly, transcriptional sex-biased genes were overrepresented among epigenetic sex-biased genes (p-value = 4.6e-13), suggesting differential DNA methylation and gene expression between male and female muscle are functionally linked. Finally, we validated expression of three genes with large effect sizes (FOXO3A, ALDH1A1, and GGT7) in the Gene SMART cohort with qPCR. GGT7, involved in antioxidant metabolism, displays male-biased expression as well as lower methylation in males across the three cohorts. In conclusion, we uncovered 8,420 genes that exhibit DNA methylation differences between males and females in human skeletal muscle that may modulate mechanisms controlling muscle metabolism and health. SignificanceThe importance of uncovering biological sex differences and their translation to physiology has become increasingly evident. Using a large-scale meta-analysis of three cohorts, we perform the first comparison of genome-wide skeletal muscle DNA methylation between males and females, and identify thousands of genes that display sex-differential methylation. We then explore intrinsic biological factors that may be underlying the DNA methylation sex differences, such as fibre type proportions and sex hormones. Leveraging the GTEx database, we identify hundreds of genes with both sex-differential expression and DNA methylation in skeletal muscle. We further confirm the sex-biased genes with gene expression data from two cohorts included in the methylation meta-analysis. Our study integrates genomewide sex-biased DNA methylation and expression in skeletal muscle, shedding light on distinct sex differences in skeletal muscle.

genomics↗