The functional consequences of age-related changes in microRNA expression in skeletal muscle.

A common characteristic of ageing is disrupted homeostasis between growth and atrophy of skeletal muscle resulting in loss of muscle mass and function, which is associated with sarcopenia. Sarcopenia is related to impaired balance, increased falls and decline in quality of life of older people. Ageing-related transcriptome and proteome changes in skeletal muscle have been characterised, however the molecular mechanisms underlying sarcopenia are still not fully understood. microRNAs are novel regulators of gene expression known to modulate skeletal muscle development and homeostasis. Expression of numerous microRNAs is disrupted in skeletal muscle with age however, the functional consequences of this are not yet understood. Given that a single microRNA can simultaneously affect multiple signalling pathways, microRNAs are potent modulators of pathophysiological changes occurring during ageing. Here we use microRNA and transcript expression profiling together with microRNA functional assays to show that disrupted microRNA:target interactions play an important role in maintaining muscle homeostasis. We identified miR-181a as a regulator of the sirtuin1 (Sirt1) gene expression in skeletal muscle and show that the expression of miR-181a and its target gene is disrupted in skeletal muscle from old mice. Moreover, we show that miR-181a:Sirt1 interactions regulate myotube size. Our results demonstrate that disrupted microRNA:target interactions are likely related to the pathophysiological changes occurring in skeletal muscle during ageing.

From Planning Stage Towards FAIR Data: A Practical Metadatasheet For Biomedical Scientists.

Datasets consist of measurement data and metadata. Metadata provides context, essential for understanding and (re-)using data. Various metadata standards exist for different methods, systems and contexts. However, relevant information resides at differing stages across the data-lifecycle. Often, this information is defined and standardized only at publication stage, which can lead to data loss and workload increase. In this study, we developed Metadatasheet, a metadata standard based on interviews with members of two biomedical consortia and systematic screening of data repositories. It aligns with the data-lifecycle allowing synchronous metadata recording within Microsoft Excel, a widespread data recording software. Additionally, we provide an implementation, the Metadata Workbook, that offers user-friendly features like automation, dynamic adaption, metadata integrity checks, and export options for various metadata standards. By design and due to its extensive documentation, the proposed metadata standard simplifies recording and structuring of metadata for biomedical scientists, promoting practicality and convenience in data management. This framework can accelerate scientific progress by enhancing collaboration and knowledge transfer throughout the intermediate steps of data creation.

miR-24 and its target gene Prdx6 regulate viability and senescence of myogenic progenitors during aging.

Satellite cell-dependent skeletal muscle regeneration declines during aging. Disruptions within the satellite cells and their niche, together with alterations in the myofibrillar environment, contribute to age-related dysfunction and defective muscle regeneration. In this study, we demonstrated an age-related decline in satellite cell viability and myogenic potential and an increase in ROS and cellular senescence. We detected a transient upregulation of miR-24 in regenerating muscle from adult mice and downregulation of miR-24 during muscle regeneration in old mice. FACS-sorted satellite cells were characterized by decreased levels of miR-24 and a concomitant increase in expression of its target: Prdx6. Using GFP reporter constructs, we demonstrated that miR-24 directly binds to its predicted site within Prdx6 mRNA. Subtle changes in Prdx6 levels following changes in miR-24 expression indicate miR-24 plays a role in fine-tuning Prdx6 expression. Changes in miR-24 and Prdx6 levels were associated with altered mitochondrial ROS generation, increase in the DNA damage marker: phosphorylated-H2Ax and changes in viability, senescence, and myogenic potential of myogenic progenitors from mice and humans. The effects of miR-24 were more pronounced in myogenic progenitors from old mice, suggesting a context-dependent role of miR-24 in these cells, with miR-24 downregulation likely a part of a compensatory response to declining satellite cell function during aging. We propose that downregulation of miR-24 and subsequent upregulation of Prdx6 in muscle of old mice following injury are an adaptive response to aging, to maintain satellite cell viability and myogenic potential through regulation of mitochondrial ROS and DNA damage pathways.

miR-181a regulates p62/SQSTM1, parkin, and protein DJ-1 promoting mitochondrial dynamics in skeletal muscle aging.

One of the key mechanisms underlying skeletal muscle functional deterioration during aging is disrupted mitochondrial dynamics. Regulation of mitochondrial dynamics is essential to maintain a healthy mitochondrial population and prevent the accumulation of damaged mitochondria; however, the regulatory mechanisms are poorly understood. We demonstrated loss of mitochondrial content and disrupted mitochondrial dynamics in muscle during aging concomitant with dysregulation of miR-181a target interactions. Using functional approaches and mito-QC assay, we have established that miR-181a is an endogenous regulator of mitochondrial dynamics through concerted regulation of Park2, p62/SQSTM1, and DJ-1 in vitro. Downregulation of miR-181a with age was associated with an accumulation of autophagy-related proteins and abnormal mitochondria. Restoring miR-181a levels in old mice prevented accumulation of p62, DJ-1, and PARK2, and improved mitochondrial quality and muscle function. These results provide physiological evidence for the potential of microRNA-based interventions for age-related muscle atrophy and of wider significance for diseases with disrupted mitochondrial dynamics.

Micro(RNA)-managing muscle wasting.

Progressive skeletal muscle wasting is a natural consequence of aging and is common in chronic and acute diseases. Loss of skeletal muscle mass and function (strength) often leads to frailty, decreased independence, and increased risk of hospitalization. Despite progress made in our understanding of the mechanisms underlying muscle wasting, there is still no treatment available, with exercise training and dietary supplementation improving, but not restoring, muscle mass and/or function. There has been slow progress in developing novel therapies for muscle wasting, either during aging or disease, partially due to the complex nature of processes underlying muscle loss. The mechanisms of muscle wasting are multifactorial, with a combination of factors underlying age- and disease-related functional muscle decline. These factors include well-characterized changes in muscle such as changes in protein turnover and more recently described mechanisms such as autophagy or satellite cell senescence. Advances in transcriptomics and other high-throughput approaches have highlighted significant deregulation of skeletal muscle gene and protein levels during aging and disease. These changes are regulated at different levels, including posttranscriptional gene expression regulation by microRNAs. microRNAs, potent regulators of gene expression, modulate many processes in muscle, and microRNA-based interventions have been recently suggested as a promising new therapeutic strategy against alterations in muscle homeostasis. Here, we review recent developments in understanding the aging-associated mechanisms of muscle wasting and explore potential microRNA-based therapeutic avenues.

Redox responses are preserved across muscle fibres with differential susceptibility to aging.

Age-related loss of muscle mass and function is associated with increased frailty and loss of independence. The mechanisms underlying the susceptibility of different muscle types to age-related atrophy are not fully understood. Reactive oxygen species (ROS) are recognised as important signalling molecules in healthy muscle and redox sensitive proteins can respond to intracellular changes in ROS concentrations modifying reactive thiol groups on Cysteine (Cys) residues. Conserved Cys residues tend to occur in functionally important locations and can have a direct impact on protein function through modifications at the active site or determining protein conformation. The aim of this work was to determine age-related changes in the redox proteome of two metabolically distinct murine skeletal muscles, the quadriceps a predominantly glycolytic muscle and the soleus which contains a higher proportion of mitochondria. To examine the effects of aging on the global proteome and the oxidation state of individual redox sensitive Cys residues, we employed a label free proteomics approach including a differential labelling of reduced and reversibly oxidised Cys residues. Our results indicate the proteomic response to aging is dependent on muscle type but redox changes that occur primarily in metabolic and cytoskeletal proteins are generally preserved between metabolically distinct tissues. BIOLOGICAL SIGNIFICANCE: Skeletal muscle containing fast twitch glycolytic fibres are more susceptible to age related atrophy compared to muscles with higher proportions of oxidative slow twitch fibres. Contracting skeletal muscle generates reactive oxygen species that are required for correct signalling and adaptation to exercise and it is also known that the intracellular redox environment changes with age. To identify potential mechanisms for the distinct response to age, this article combines a global proteomic approach and a differential labelling of reduced and reversibly oxidised Cysteine residues in two metabolically distinct skeletal muscles, quadriceps and soleus, from adult and old mice. Our results indicate that the global proteomic changes with age in skeletal muscles are dependent on fibre type. However, redox specific changes are preserved across muscle types and accompanied with a reduction in the number of redox sensitive Cysteine residues.

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans.

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting occurs. Loss of muscle mass and function is associated with muscle fiber type atrophy, fiber type switching, defective muscle regeneration associated with dysfunction of satellite cells, muscle stem cells, and other pathophysiological processes. These changes are associated with changes in intracellular as well as local and systemic niches. In addition to most commonly used rodent models of muscle ageing, there is a need to study muscle homeostasis and wasting using human models, which due to ethical implications, consist predominantly of in vitro cultures. Despite the wide use of human Myogenic Progenitor Cells (MPCs) and primary myoblasts in myogenesis, there is limited data on using human primary myoblast and myotube cultures to study molecular mechanisms regulating different aspects of age-associated muscle wasting, aiding in the validation of mechanisms of ageing proposed in rodent muscle. The use of human MPCs, primary myoblasts and myotubes isolated from adult and aged people, provides a physiologically relevant model of molecular mechanisms of processes associated with muscle growth, atrophy and regeneration. Here we describe in detail a robust, inexpensive, reproducible and efficient protocol for the isolation and maintenance of human MPCs and their progeny - myoblasts and myotubes from human muscle samples using enzymatic digestion. Furthermore, we have determined the passage number at which primary myoblasts from adult and aged people undergo senescence in an in vitro culture. Finally, we show the ability to transfect these myoblasts and the ability to characterize their proliferative and differentiation capacity and propose their suitability for performing functional studies of molecular mechanisms of myogenesis and muscle wasting in vitro.

Age-related changes in miR-143-3p:Igfbp5 interactions affect muscle regeneration.

A common characteristic of aging is defective regeneration of skeletal muscle. The molecular pathways underlying age-related decline in muscle regenerative potential remain elusive. microRNAs are novel gene regulators controlling development and homeostasis and the regeneration of most tissues, including skeletal muscle. Here, we use satellite cells and primary myoblasts from mice and humans and an in vitro regeneration model, to show that disrupted expression of microRNA-143-3p and its target gene, Igfbp5, plays an important role in muscle regeneration in vitro. We identified miR-143 as a regulator of the insulin growth factor-binding protein 5 (Igfbp5) in primary myoblasts and show that the expression of miR-143 and its target gene is disrupted in satellite cells from old mice. Moreover, we show that downregulation of miR-143 during aging may act as a compensatory mechanism aiming at improving myogenesis efficiency; however, concomitant upregulation of miR-143 target gene, Igfbp5, is associated with increased cell senescence, thus affecting myogenesis. Our data demonstrate that dysregulation of miR-143-3p:Igfbp5 interactions in satellite cells with age may be responsible for age-related changes in satellite cell function.