Biology subjects

Hildyard, J. C. W.

Publications and source records attributed to Hildyard, J. C. W..

7 recordsLinked to original sources

bioRxiv2026

A positively selected microRNA controls a reversible aging program in striated muscle

Aging is the primary risk factor for most chronic diseases and is characterized in striated muscle by progressive functional decline, mitochondrial dysfunction, and chronic inflammation. The miR-128-1 locus resides within a positively selected haplotype on chromosome 2q21.3 associated with variation in grip strength, pulmonary function, and cardiometabolic traits in humans. Here, we show that antisense oligonucleotide-mediated inhibition of miR-128-3p restores muscle mass and function in aged mice, improves cardiac function while limiting adverse remodeling following myocardial infarction, and ameliorates skeletal and cardiac muscle pathology in mouse and pig models of Duchenne muscular dystrophy. Across these contexts, miR-128-3p inhibition induces a conserved transcriptional response characterized by activation of mitochondrial programs and suppression of inflammatory and fibrotic signaling, resembling the effects of established longevity interventions. These findings identify miR-128-3p as a regulator of a conserved aging-associated program and establish its inhibition as a strategy to restore tissue function across aging-related muscle pathologies.

molecular biology↗

bioRxiv2025

Myonuclear domain-associated and central nucleation-dependent spatial restriction of dystrophin protein expression in a novel DMD mouse model

The restoration of uniformly-distributed dystrophin protein expression is an important consideration for the development of advanced therapeutics for Duchenne muscular dystrophy (DMD). To explore this concept, we generated a novel genetic mouse model (mdx52-Xist{Delta}hs) that expresses variable, and non-uniformly distributed, dystrophin protein from birth as a consequence of skewed X-chromosome inactivation. mdx52-Xist{Delta}hs myofibers are heterokaryons containing a mixture of myonuclei expressing either wild-type or mutant dystrophin alleles in a mutually exclusive manner, resulting in dystrophin protein being spatially restricted to corresponding dystrophin-expressing myonuclear domains. This phenotype models the situation in female DMD carriers, and dystrophic muscle in which dystrophin has been incompletely restored by partially-effective experimental therapeutics. Total dystrophin expression increased in aged (60-week-old) mdx52-Xist{Delta}hs mice relative to 6-week-old adults, suggestive of an accumulation of dystrophin-expressing myonuclei through positive selection, although this was insufficient to resolve sarcolemmal dystrophin patchiness. Nevertheless, compared to mice expressing no dystrophin, non-uniformly-distributed dystrophin was protective against pathology-related muscle turnover in an expression-level-dependent manner in both adult and aged mdx52-Xist{Delta}hs mice. Systematic classification of isolated mdx52-Xist{Delta}hs myofibers revealed profound differences associated with central nucleation, with dystrophin found to be translationally repressed in centrally-nucleated myofibers and myofiber segments. These findings have important implications for the development of dystrophin restoration therapies.

cell biology↗

bioRxiv2024

Spatiotemporal analysis of dystrophin expression during muscle repair

Dystrophin mRNA is produced from a very large genetic locus and transcription of a single mRNA requires approximately 16 hours. This prolonged interval between transcriptional initiation and completion results in unusual transcriptional behaviour: in skeletal muscle, myonuclei express dystrophin continuously and robustly, yet degrade mature transcripts shortly after completion, such that most dystrophin mRNA is nascent, not mature. This implies dystrophin expression is principally controlled post-transcriptionally, a mechanism that circumvents transcriptional delay, allowing rapid responses to change in demand. Dystrophin protein is however highly stable, with slow turnover: in healthy muscle, despite constant production of dystrophin mRNA, demand is low and the need for responsive expression is minimal. We reasoned this system instead exists to control dystrophin expression during rare periods of elevated but changing demand, such as during muscle development or repair, when newly formed fibres must establish sarcolemmal dystrophin rapidly. By assessing dystrophin mRNA and protein expression in regenerating skeletal muscle following injury, we reveal a complex program that suggests control at multiple levels: nascent transcription begins even prior to myoblast fusion, effectively paying in advance to minimise subsequent delay. During myotube differentiation and maturation, when sarcolemmal demands are high, initiation increases only modestly while mature transcript stability increases markedly to generate high numbers of mature dystrophin transcripts, a state that persists until repair is complete, when a state of oversupply and degradation resumes. Our data demonstrate that dystrophin mRNA is indeed chiefly controlled by turnover, not initiation: degradation consequently represents a potential therapeutic target for maximising efficacy of even modest dystrophin restoration.

cell biology↗

bioRxiv2024

Evaluation of suitable reference genes for qPCR normalisation of gene expression in a Achilles tendon injury model.

Tendons are one of the major load-bearing tissues in the body; subjected to enormous peak stresses, and thus vulnerable to injury. Cellular responses to tendon injury are complex, involving inflammatory and repair components, with the latter employing both resident and recruited exogenous cell populations. Gene expression analyses are valuable tools for investigating tendon injury, allowing assessment of repair processes and pathological responses such as fibrosis, and permitting evaluation of therapeutic pharmacological interventions. Quantitative polymerase chain reaction (qPCR) is a commonly used approach for such studies, but data obtained by this method must be normalised to reference genes: genes known to be stably expressed between the experimental conditions investigated. Establishing suitable tendon injury reference genes is thus essential. Accordingly we investigated mRNA expression stability in a rat model of tendon injury, comparing both injured and uninjured tendons, and the effects of rapamycin treatment, at 1 and 3 weeks post injury. We used 11 candidate genes (18S, ACTB, AP3D1, B2M, CSNK2A2, GAPDH, HPRT1, PAK1IP1, RPL13a, SDHA, UBC) and assessed stability via four complementary algorithms (Bestkeeper, deltaCt, geNorm, Normfinder). Our results suggests that ACTB, CSNK2A2, HPRT1 and PAK1IP1 are all stably expressed in tendon, regardless of injury or drug treatment: any three of these would serve as universally suitable reference gene panel for normalizing qPCR expression data in the rat tendon injury model. We also reveal 18S, UBC, GAPDH, and SDHA as consistently poor scoring candidates (with the latter two exhibiting rapamycin- and injury-associated changes, respectively): these genes should be avoided.

molecular biology↗

bioRxiv2022

Rapid histological quantification of muscle fibrosis and lysosomal activity using the HSB colour space

IntroductionFibrosis is a key feature of many chronic myopathic disorders, such as in the muscle-wasting condition, Duchenne muscular dystrophy. Fibrosis disrupts skeletal muscle architecture, limits muscle function, impairs regeneration and might reduce efficacy of therapeutic interventions: quantifying muscle fibrosis is thus of key value in monitoring disease progression (or response to treatment) in both pre-clinical and clinical settings. Fibrosis can be visualised histologically via staining with picrosirius red, but its quantification can be time consuming and subject to investigator bias: a rapid, reliable and user-friendly means of quantifying muscle fibrosis in histological images is currently lacking. MethodsWe investigated whether the Hue/Saturation/Brightness (HSB) colour-space could be used to quantify connective tissue content in picrosirius red (PSR)-stained muscle sections, using multiple healthy and dystrophic muscles, sampled from two animal models of Duchenne muscular dystrophy (the mdx mouse and the DE50-MD dog), at different ages. ResultsHSB-based analysis allows muscle fibres, connective tissue and slide background to be readily distinguished in PSR images using only a minimal set of parameters, and correctly identifies fibrotic accumulation under conditions where progressive fibrosis is expected. We have developed an imageJ macro that allows semi-automated high-throughput measurement of fibrotic accumulation, and then further extended our method to demonstrate its validity with another histological stain (acid phosphatase) to quantify lysosomal activity. ConclusionsHistological analysis of muscle pathology is challenging and time consuming, especially with large collections of images: our methods permit fibrotic accumulation to be quantified in such collections rapidly and easily in open-source software, with minimal hardware requirements, and the underlying methodology can be readily extended to other colorimetric histopathological stains.

molecular biology↗

bioRxiv2020

Identification of qPCR reference genes suitable for normalising gene expression in the developing mouse embryo

Mammalian embryogenesis is an intricate, tightly orchestrated process. Progression from zygote through somitogenesis and on to organogenesis and maturity involves many interacting cell types and multiple differentiating cell lineages. Quantitative PCR analysis of gene expression in the developing embryo is a valuable tool for deciphering these interactions and tracing lineages, but normalisation of qPCR data to stably expressed reference genes is essential. Patterns of gene expression change globally and dramatically as embryonic development proceeds, rendering identification of appropriate reference genes challenging at both the whole embryo- and individual tissue-level. We have investigated expression stability in mouse embryos from mid to late gestation (E11.5-E18.5), both at the whole-embryo level, and within more restricted tissue domains (head, developing forelimb), using 15 candidate reference genes (ACTB, 18S, SDHA, GAPDH, HTATSF1, CDC40, RPL13A, CSNK2A2, AP3D1, HPRT1, CYC1, EIF4A, UBC, B2M and PAK1IP1), and four complementary algorithms (geNorm, Normfinder, Bestkeeper and deltaCt). Unexpectedly, all methods suggest that many genes within our candidate panel are acceptable references, and despite disagreement over highest-scoring candidates, AP3D1, RPL14A and PAK1IP1 are the strongest performing genes overall. Conversely, HPRT1 and B2M are consistently poor choices: these genes show strong developmental regulation. We further show that use of AP3D1, RPL13A and PAK1IP1 can reveal subtle patterns of developmental expression even in genes ostensibly ranked as acceptable (CDC40, HTATSF1), and thus these three represent universally suitable reference genes for the mouse embryo.

molecular biology↗

bioRxiv2019

Multiplex in situ hybridization within a single transcript: RNAscope reveals dystrophin mRNA dynamics

Dystrophin plays a vital role in maintaining muscle health, yet low mRNA expression, lengthy transcription time and the limitations of traditional in-situ hybridization (ISH) methodologies mean that the dynamics of dystrophin transcription remain poorly understood. RNAscope is highly sensitive ISH method that can be multiplexed, allowing detection of individual transcripts at sub-cellular resolution, with different target mRNAs assigned to distinct fluorophores. We present a novel approach, instead using RNAscope probes targeted to 5 and 3 regions of the same transcript: labelling muscle dystrophin mRNA in this manner allows transcriptional dynamics to be deciphered in health and disease, resolving both nascent myonuclear transcripts and exported mature mRNAs (the latter absent in dystrophic muscle, yet restored following therapeutic intervention). We show that even in healthy muscle, immature dystrophin mRNA predominates (60-80% of total), with the surprising implication that the half-life of a mature transcript is markedly shorter than the time invested in transcription: at the transcript level, supply may exceed demand. Our findings provide unique spatiotemporal insight into the behaviour of this long transcript (with implications for therapeutic approaches), and further suggests this modified multiplex ISH approach is well-suited to long genes, offering a highly tractable means to reveal complex transcriptional dynamics.

cell biology↗