Katarzyna Goljanek-Whysall

MicroRNA regulation of the paired-box transcription factor Pax3 confers robustness to developmental timing of myogenesis

Commitment of progenitors in the dermomyotome to myoblast fate is the first step in establishing the body musculature. Pax3 is a crucial transcription factor, important for skeletal muscle development and expressed in myogenic progenitors in the dermomyotome of developing somites and in migratory muscle progenitors that populate the limb buds. Down-regulation of Pax3 is essential to ignite the myogenic program, including up-regulation of myogenic regulators, Myf-5 and MyoD. MicroRNAs (miRNAs) confer robustness to developmental timing by posttranscriptional repression of genetic programs that are related to previous developmental stages or to alternative cell fates. Here we demonstrate that the muscle-specific miRNAs miR-1 and miR-206 directly target Pax3. Antagomir-mediated inhibition of miR-1/miR-206 led to delayed myogenic differentiation in developing somites, as shown by transient loss of myogenin expression. This correlated with increased Pax3 and was phenocopied using Pax3-specific target protectors. Loss of myogenin after antagomir injection was rescued by Pax3 knockdown using a splice morpholino, suggesting that miR-1/miR-206 control somite myogenesis primarily through interactions with Pax3. Our studies reveal an important role for miR-1/miR-206 in providing precision to the timing of somite myogenesis. We propose that posttranscriptional control of Pax3 downstream of miR-1/miR-206 is required to stabilize myoblast commitment and subsequent differentiation. Given that mutually exclusive expression of miRNAs and their targets is a prevailing theme in development, our findings suggest that miRNA may provide a general mechanism for the unequivocal commitment underlying stem cell differentiation.

microRNAs in skeletal muscle differentiation and disease.

miRNAs (microRNAs) are novel post-transcriptional regulators of gene expression. Several miRNAs, expressed exclusively in muscle, play important roles during muscle development, growth and regeneration; other ubiquitously expressed miRNAs are also essential for muscle function. In the present review, we outline the miRNAs involved in embryonic muscle development and those that have been found to be dysregulated in diseases associated with skeletal muscle or are changed during muscle adaptation. miRNAs are promising biomarkers and candidates for potential therapeutic intervention. We discuss the strategies that aim to develop novel therapies through modulating miRNA activity. In time, some of these approaches may become available to treat muscle-associated diseases.

myomiR-dependent switching of BAF60 variant incorporation into Brg1 chromatin remodeling complexes during embryo myogenesis

Myogenesis involves the stable commitment of progenitor cells followed by the execution of myogenic differentiation, processes that are coordinated by myogenic regulatory factors, microRNAs and BAF chromatin remodeling complexes. BAF60a, BAF60b and BAF60c are structural subunits of the BAF complex that bind to the core ATPase Brg1 to provide functional specificity. BAF60c is essential for myogenesis; however, the mechanisms regulating the subunit composition of BAF/Brg1 complexes, in particular the incorporation of different BAF60 variants, are not understood. Here we reveal their dynamic expression during embryo myogenesis and uncover the concerted negative regulation of BAF60a and BAF60b by the muscle-specific microRNAs (myomiRs) miR-133 and miR-1/206 during somite differentiation. MicroRNA inhibition in chick embryos leads to increased BAF60a or BAF60b levels, a concomitant switch in BAF/Brg1 subunit composition and delayed myogenesis. The phenotypes are mimicked by sustained BAF60a or BAF60b expression and are rescued by morpholino knockdown of BAF60a or BAF60b. This suggests that myomiRs contribute to select BAF60c for incorporation into the Brg1 complex by specifically targeting the alternative variants BAF60a and BAF60b during embryo myogenesis, and reveals that interactions between tissue-specific non-coding RNAs and chromatin remodeling factors confer robustness to mesodermal lineage determination.

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.

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.

microRNAs: Modulators of the underlying pathophysiology of sarcopenia?

Skeletal muscle homeostasis depends on an intricate balance between muscle hypertrophy, atrophy and regeneration. As we age, maintenance of muscle homeostasis is perturbed, resulting in a loss of muscle mass and function, termed sarcopenia. Individuals with sarcopenia exhibit impaired balance, increased falls (leading to subsequent injury) and an overall decline in quality of life. The mechanisms mediating sarcopenia are still not fully understood but clarity in our understanding of the precise pathophysiological changes occurring during skeletal muscle ageing has improved dramatically. Advances in transcriptomics has highlighted significant deregulation in skeletal muscle gene expression with ageing, suggesting epigenetic alterations may play a crucial and potentially causative role in the skeletal muscle ageing process. microRNAs (miRNAs, miRs), novel regulators of gene expression, can modulate many processes in skeletal muscle, including myogenesis, tissue regeneration and cellular programming. Expression of numerous evolutionary conserved miRNAs is disrupted in skeletal muscle with age. Given that a single miRNA can simultaneously affect the functionality of multiple signaling pathways, miRNAs are potent modulators of pathophysiological changes. miRNA-based interventions provide a promising new therapeutic strategy against alterations in muscle homeostasis. The aim of this review is two-fold; firstly to outline the latest understanding of the pathophysiological alterations impacting the deregulation of skeletal muscle mass and function with ageing, and secondly, to highlight the mounting evidence for a role of miRNAs in modulating muscle mass, and the need to explore their specific role in sarcopenia.

Decoding the Regulatory Landscape of Ageing in Musculoskeletal Engineered Tissues Using Genome-Wide DNA Methylation and RNASeq

Mesenchymal stem cells (MSC) are capable of multipotent differentiation into connective tissues and as such are an attractive source for autologous cell-based regenerative medicine and tissue engineering. Epigenetic mechanisms, like DNA methylation, contribute to the changes in gene expression in ageing. However there was a lack of sufficient knowledge of the role that differential methylation plays during chondrogenic, osteogenic and tenogenic differentiation from ageing MSCs. This study undertook genome level determination of the effects of DNA methylation on expression in engineered tissues from chronologically aged MSCs. We compiled unique DNA methylation signatures from chondrogenic, osteogenic, and tenogenic engineered tissues derived from young; n = 4 (21.8 years ± 2.4 SD) and old; n = 4 (65.5 years±8.3SD) human MSCs donors using the Illumina HumanMethylation 450 Beadchip arrays and compared these to gene expression by RNA sequencing. Unique and common signatures of global DNA methylation were identified. There were 201, 67 and 32 chondrogenic, osteogenic and tenogenic age-related DE protein-coding genes respectively. Findings inferred the nature of the transcript networks was predominantly for ‘cell death and survival’, ‘cell morphology’, and ‘cell growth and proliferation’. Further studies are required to validate if this gene expression effect translates to cell events. Alternative splicing (AS) was dysregulated in ageing with 119, 21 and 9 differential splicing events identified in chondrogenic, osteogenic and tenogenic respectively, and enrichment in genes associated principally with metabolic processes. Gene ontology analysis of differentially methylated loci indicated age-related enrichment for all engineered tissue types in ‘skeletal system morphogenesis’, ‘regulation of cell proliferation’ and ‘regulation of transcription’ suggesting that dynamic epigenetic modifications may occur in genes associated with shared and distinct pathways dependent upon engineered tissue type. An altered phenotype in engineered tissues was observed with ageing at numerous levels. These changes represent novel insights into the ageing process, with implications for stem cell therapies in older patients. In addition we have identified a number of tissue-dependant pathways, which warrant further studies.

Ageing in relation to skeletal muscle dysfunction: redox homoeostasis to regulation of gene expression

Ageing is associated with a progressive loss of skeletal muscle mass, quality and function—sarcopenia, associated with reduced independence and quality of life in older generations. A better understanding of the mechanisms, both genetic and epigenetic, underlying this process would help develop therapeutic interventions to prevent, slow down or reverse muscle wasting associated with ageing. Currently, exercise is the only known effective intervention to delay the progression of sarcopenia. The cellular responses that occur in muscle fibres following exercise provide valuable clues to the molecular mechanisms regulating muscle homoeostasis and potentially the progression of sarcopenia. Redox signalling, as a result of endogenous generation of ROS/RNS in response to muscle contractions, has been identified as a crucial regulator for the adaptive responses to exercise, highlighting the redox environment as a potentially core therapeutic approach to maintain muscle homoeostasis during ageing. Further novel and attractive candidates include the manipulation of microRNA expression. MicroRNAs are potent gene regulators involved in the control of healthy and disease-associated biological processes and their therapeutic potential has been researched in the context of various disorders, including ageing-associated muscle wasting. Finally, we discuss the impact of the circadian clock on the regulation of gene expression in skeletal muscle and whether disruption of the peripheral muscle clock affects sarcopenia and altered responses to exercise. Interventions that include modifying altered redox signalling with age and incorporating genetic mechanisms such as circadian- and microRNA-based gene regulation, may offer potential effective treatments against age-associated sarcopenia.

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.

MicroRNA Dysregulation in Aging and Pathologies of the Skeletal Muscle

Skeletal muscle is one of the biggest organs of the body with important mechanistic and metabolic functions. Muscle homeostasis is controlled by environmental, genetic, and epigenetic factors. Indeed, MiRNAs, small noncoding RNAs robust regulators of gene expression, have and have been shown to regulate muscle homeostasis on several levels: through controlling myogenesis, muscle growth (hypertrophy) and atrophy, as well as interactions of muscle with other tissues. Given the large number of MiRNA target genes and the important role of MiRNAs in most physiological processes and various diseases, MiRNAs may have an enormous potential as therapeutic targets against numerous disorders, including pathologies of muscle. The purpose of this review is to present the current knowledge of the role of MiRNAs in skeletal muscle homeostasis and pathologies and the potential of MiRNAs as therapeutics for skeletal muscle wasting, with particular focus on the age- and disease-related loss of muscle mass and function.