Biology subjects

Wadley, G. D.

Publications and source records attributed to Wadley, G. D..

6 recordsLinked to original sources

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↗

bioRxiv2025

Skeletal muscle mitochondria contain nuclear-encoded RNA species prior to and following adaptation to exercise training in rats

Skeletal muscle mitochondria adaptation to exercise training is mediated by molecular factors that are not fully understood. Mitochondria import over 1000 proteins encoded by the nuclear genome, but the RNA population resident within the organelle is generally thought to be exclusively encoded by the mitochondrial genome. However, recent in vitro evidence suggests that specific nuclear-encoded miRNAs and other non-coding RNAs (ncRNAs) can reside within the mitochondrial matrix. Whether these are present in mitochondria of skeletal muscle tissue, and if this affected by endurance training - a potent metabolic stimulus for mitochondrial adaptation - remains unknown. Rats underwent four weeks of moderate intensity treadmill exercise training, then humanely killed and tissues collected for molecular profiling. Mitochondria from gastrocnemius skeletal muscle were isolated by immunoprecipitation, further purified, then the resident RNA was sequenced to assess the mitochondrial transcriptome. Exercise training elicited typical transcriptomic responses and functional adaptations in skeletal muscle, including increased mitochondrial respiratory capacity. We identified 24 nuclear-encoded, coding or non-coding RNAs in purified mitochondria, in addition to 50 nuclear-encoded miRNAs that met a specified abundance threshold. Although none were differentially expressed in the exercise vs control group at FDR<0.05, exploratory analyses suggested that the abundance of 3 miRNAs were altered (p<0.05) in mitochondria isolated from trained compared with sedentary skeletal muscle. We report the presence of a specific population of nuclear-encoded RNAs in the mitochondria isolated from rat skeletal muscle tissue, which could play a role in regulating exercise adaptations and mitochondrial biology. KEY POINTSO_LIMitochondria are a central metabolic and signalling hub, particularly in tissues with high energy demand such as skeletal muscle. Their ability to adapt to stimuli such as exercise training is directly related to improved metabolic health. Understanding the factors that regulate these adaptive processes is therefore essential. C_LIO_LIMitochondria contain RNA encoded by their own genome (mtDNA); typically are not thought to contain nuclear-encoded RNA. C_LIO_LIHere, we report that mitochondria from rat skeletal muscle do indeed contain a select population of nuclear-encoded protein coding and non-coding RNAs, including 3 microRNAs whose expression tended to be altered by exercise training. C_LIO_LIThese findings suggest that mitochondria-localised nuclear-encoded RNAs may play a role in mediating beneficial adaptive responses to exercise. 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↗

bioRxiv2023

Regulation of mitochondrial calcium uniporter expression and calcium signalling by lncRNA Tug1 in cardiomyocytes.

Cardiomyocyte calcium homeostasis is a tightly regulated process. The mitochondrial calcium uniporter (MCU) complex can buffer elevated cytosolic Ca2+ levels and consists of pore-forming proteins including MCU, and various regulatory proteins such as mitochondrial calcium uptake proteins 1 and 2 (MICU1/2). The stoichiometry of these proteins influences the sensitivity to Ca2+ and activity of the complex. However, the factors that regulate their gene expression remain incompletely understood. Long non-coding RNAs (lncRNAs) regulate gene expression through various mechanisms, and we recently found that the lncRNA Tug1 increased the expression of Mcu and associated genes. To further explore this, we performed antisense LNA knockdown of Tug1 (Tug1 KD) in H9c2 rat cardiomyocytes. Tug1 KD increased MCU protein expression, yet pyruvate dehydrogenase dephosphorylation, which is indicative of mitochondrial Ca2+ uptake was not enhanced. However, RNA-seq revealed that Tug1 KD increased Mcu along with differential expression of >1000 genes including many related to Ca2+ regulation pathways in the heart. To understand the effect of this on Ca2+ signalling, we measured phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and its downstream target cAMP Response Element-Binding protein (CREB), a transcription factor known to drive Mcu gene expression. In response a Ca2+ stimulus, the increase in CaMKII and CREB phosphorylation was attenuated by Tug1 KD. Inhibition of CaMKII, but not CREB, partially prevented the Tug1 KD- mediated increase in Mcu. Together, these data suggest that Tug1 modulates MCU expression via a mechanism involving CaMKII and regulates cardiomyocyte Ca2+ signalling which could have important implications for cardiac function.

cell biology↗

bioRxiv2022

Purification of mitochondria from skeletal muscle tissue for transcriptomic analyses reveals localisation of nuclear-encoded non-coding RNAs

Mitochondria are central to cellular function, particularly in metabolically active tissues such as skeletal muscle. Nuclear-encoded RNAs typically localise within the nucleus and cytosol but a small population may also translocate to subcellular compartments such as mitochondria. We aimed to investigate the nuclear-encoded RNAs that localise within the mitochondria of skeletal muscle cells and tissue. Intact mitochondria were isolated via immunoprecipitation (IP) followed by enzymatic treatments (RNase-A and proteinase-K) optimised to remove transcripts located exterior to mitochondria, making it amenable for high-throughput transcriptomic sequencing. Small-RNA sequencing libraries were successfully constructed from as little as 1.8 ng mitochondrial RNA input. Small-RNA sequencing of mitochondria from rat myoblasts revealed the enrichment of over 200 miRNAs. Whole-transcriptome RNA sequencing of enzymatically-purified mitochondria isolated by IP from skeletal muscle tissue showed a striking similarity in the degree of purity compared to mitoplast preparations which lack an outer mitochondrial membrane. In summary, we describe a novel, powerful sequencing approach applicable to animal and human tissues and cells that can facilitate the discovery of nuclear-encoded RNA transcripts localised within skeletal muscle mitochondria.

cell biology↗