Biology subjects

Trewin, A. J.

Publications and source records attributed to Trewin, A. J..

7 recordsLinked to original sources

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

Sex-specific regulation of the cardiac transcriptome by the protein phosphatase 2A regulatory subunit B55α

1.Protein phosphatase 2A (PP2A) regulatory subunit B55 has been implicated in the transcriptional regulation of cardiac growth and fibrosis by suppressing HDAC5/MEF2 signalling in cardiomyocytes. We created and characterised two mouse models with global or cardiomyocyte-specific disruption of the gene encoding B55 (Ppp2r2a) to conduct the first detailed exploration of B55 in the heart. Global homozygous B55 knockout mice died in utero, while heterozygous mice had thinner left ventricular walls at 12 months, an effect more pronounced in males. At 10-12 weeks of age, cardiomyocyte-specific B55 knockout mice displayed normal cardiac morphology with increased left ventricular collagen deposition, identifying B55 as a negative regulator of cardiac fibrosis. Gene expression analyses revealed extensive remodelling of the cardiac transcriptome in male but not female mice, identifying a sexually dimorphic role for B55 in cardiac transcriptional regulation. These findings provide a basis for future work investigating B55 in cardiac stress settings.

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

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↗

bioRxiv2019

Quantification of reactive oxygen species production by the red fluorescent proteins KillerRed, SuperNova and mCherry.

Fluorescent proteins can generate reactive oxygen species (ROS) upon absorption of photons via type I and II photosensitization mechanisms. The red fluorescent proteins KillerRed and SuperNova are phototoxic proteins engineered to generate ROS and are used in a variety of biological applications. However, their relative quantum yields and rates of ROS production are unclear, which has limited the interpretation of their effects when used in biological systems. We cloned and purified KillerRed, SuperNova, and mCherry - a related red fluorescent protein not typically considered a photosensitizer - and measured the superoxide (O2 *-) and singlet oxygen (1O2) quantum yields with irradiation at 561 nm. The formation of the O2 *--specific product 2-hydroxyethidium (2-OHE+) was quantified via HPLC separation with fluorescence detection. Relative to a reference photosensitizer, Rose Bengal, the O2 *- quantum yield ({Phi}O2 *-) of SuperNova was determined to be 0.00150, KillerRed was 0.00097, and mCherry 0.00120. At an excitation fluence of 916.5 J/cm2 and matched absorption at 561 nm, SuperNova, KillerRed and mCherry made 3.81, 2.38 and 1.65 M O2 *-/min, respectively. Using the probe Singlet Oxygen Sensor Green (SOSG), we ascertained the 1O2 quantum yield ({Phi}1O2) for SuperNova to be 0.0220, KillerRed 0.0076, and mCherry 0.0057. These photosensitization characteristics of SuperNova, KillerRed and mCherry improve our understanding of fluorescent proteins and are pertinent for refining their use as tools to advance our knowledge of redox biology.\n\nGRAPHICAL ABSTRACT\n\nO_FIG O_LINKSMALLFIG WIDTH=183 HEIGHT=200 SRC=\"FIGDIR/small/777417v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (20K):\norg.highwire.dtl.DTLVardef@197ad04org.highwire.dtl.DTLVardef@dffa05org.highwire.dtl.DTLVardef@9765e4org.highwire.dtl.DTLVardef@1a26545_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2019

Controlling the Mitochondrial Protonmotive Force with Light to Impact Cellular Stress Resistance

Mitochondrial respiration generates an electrochemical proton gradient across the mitochondrial inner membrane called the protonmotive force (PMF) to drive diverse functions and make ATP. Current techniques to manipulate the PMF are limited to its dissipation; there is no precise, reversible method to increase the PMF. To address this issue, we used an optogenetic approach and engineered a mitochondria-targeted light-activated proton pumping protein we called mitochondria-ON (mtON) to selectively increase the PMF. Here, mtON increased the PMF light dose-dependently, supported ATP synthesis, increased resistance to mitochondrial toxins, and modulated energy-sensing behavior in Caenorhabditis elegans. Moreover, transient mtON activation during hypoxia prevented the well-characterized adaptive response of hypoxic preconditioning. Our novel optogenetic approach demonstrated that a decreased PMF is both necessary and sufficient for hypoxia-stimulated stress resistance. Our results show that optogenetic manipulation of the PMF is a powerful tool to modulate metabolic and cell signaling outcomes.

physiology↗