microRNAs In Skeletal Muscle Research Articles

Exercise Mode Combined with Essential Amino Acid and Carbohydrate Supplementation Differentially Regulate Skeletal Muscle microRNA

Skeletal muscle microRNAs (myomiR) have been implicated in modulating intracellular regulation of muscle protein synthesis by negative inhibition of the mTORC1 pathway. The acute effects of endurance exercise mode and recovery essential amino acid and carbohydrate (EAA+CHO) nutrition on myomiR expression are not well defined. PURPOSE: Determine the effects of endurance exercise mode, with or without EAA+CHO ingestion on myomiR expression. METHODS: Twenty five adults (mean ± SD; 22 ± 2 y, 82 ± 11 kg, VO2peak 4.0 ± 0.5 L[BULLET OPERATOR]min-1) performed 90 min of metabolically-matched (2.2 ± 0.1 VO2 L[BULLET OPERATOR]m-1) load carriage (LC; performed on a treadmill wearing a vest equal to 30% of individual body mass; load carried 24 ± 3 kg) or cycle ergometry (CE) exercise, during which EAA+CHO (10 g EAA and 46 g CHO) or non-nutritive control (CON) drinks were consumed. Expressions of myomiR were determined using RT-qPCR in muscle samples obtained at rest (PRE), immediately post-exercise (POST) and after 3-hr recovery (REC). RESULTS: Relative to PRE, POST and REC expressions of miR-1-3p, miR-206, miR-208a-5 and miR-499 were lower (P < 0.05) for LC compared to CE, regardless of dietary treatment. Independent of exercise mode, miR-1-3p and miR-208a-5p expression were lower (P < 0.05) after ingesting EAA+CHO compared to CON. Expression of miR-206 was highest for CE+CON than any other treatment (exercise-by-drink, P < 0.05). CONCLUSIONS: These data show that myomiR expression is differentially regulated by endurance exercise mode and EAA+CHO nutrition. Weight bearing exercise downregulates myomiR expression, whereas myomiR expression appears to be upregulated after non-weight bearing exercise. Consuming EAA+CHO attenuated the increase in myomiR expression with non-weight bearing exercise, yet the suppression of myomiR expression with feeding was more pronounced when EAA+CHO were consumed during weight bearing exercise. These findings suggest that combining weight bearing exercise with protein and carbohydrate supplementation may facilitate muscle anabolic adaptations to exercise by lowering mTORC1 inhibition.

MicroRNA in Skeletal Muscle: Its Crucial Roles in Signal Proteins, Mus cle Fiber Type, and Muscle Protein Synthesis.

Pork is one of the most economical sources of animal protein for human consumption. Meat quality is an important economic trait for the swine industry, which is primarily determined by prenatal muscle development and postnatal growth. Identification of the molecular mechanisms underlying skeletal muscle development is a key priority. MicroRNAs (miRNAs) are a class of small noncoding RNAs that have emerged as key regulators of skeletal muscle development. A number of muscle-related miRNAs have been identified by functional gain and loss experiments in mouse model. However, determining miRNA-mRNA interactions involved in pig skeletal muscle still remains a significant challenge. For a comprehensive understanding of miRNA-mediated mechanisms underlying muscle development, miRNAome analyses of pig skeletal muscle have been performed by deep sequencing. Additionally, porcine miRNA single nucleotide polymorphisms have been implicated in muscle fiber types and meat quality. The present review provides an overview of current knowledge on recently identified miRNAs involved in myogenesis, muscle fiber type and muscle protein metabolism. Undoubtedly, further systematic understanding of the functions of miRNAs in pig skeletal muscle development will be helpful to expand the knowledge of basic skeletal muscle biology and be beneficial for the genetic improvement of meat quality traits.

Skeletal Muscle myomiR Are Differentially Expressed by Endurance Exercise Mode and Combined Essential Amino Acid and Carbohydrate Supplementation

Skeletal muscle microRNAs (myomiR) expression is modulated by exercise, however, the influence of endurance exercise mode, combined with essential amino acid and carbohydrate (EAA+CHO) supplementation are not well defined. This study determined the effects of weighted versus non-weighted endurance exercise, with or without EAA+CHO ingestion on myomiR expression and their association with muscle protein synthesis (MPS). Twenty five adults performed 90 min of metabolically-matched (2.2 VO2 L·m−1) load carriage (LC; performed on a treadmill wearing a vest equal to 30% of individual body mass) or cycle ergometry (CE) exercise, during which EAA+CHO (10 g EAA and 46 g CHO) or non-nutritive control (CON) drinks were consumed. Expression of myomiR (RT-qPCR) were determined at rest (PRE), immediately post-exercise (POST), and 3 h into recovery (REC). Muscle protein synthesis (2H5-phenylalanine) was measured during exercise and recovery. Relative to PRE, POST, and REC expression of miR-1-3p, miR-206, miR-208a-5, and miR-499 was lower (P < 0.05) for LC compared to CE, regardless of dietary treatment. Independent of exercise mode, miR-1-3p and miR-208a-5p expression were lower (P < 0.05) after ingesting EAA+CHO compared to CON. Expression of miR-206 was highest for CE-CON than any other treatment (exercise-by-drink, P < 0.05). Common targets of differing myomiR were identified as markers within mTORC1 signaling, and miR-206 and miR-499 were inversely associated with MPS rates immediately post-exercise. These findings suggest the alterations in myomiR expression between exercise mode and EAA+CHO intake may in part be due to differing MPS modulation immediately post-exercise.

Exercise Altered the Skeletal Muscle MicroRNAs and Gene Expression Profiles in Burn Rats With Hindlimb Unloading.

This study investigated microRNA and target gene profiles under different conditions of burn, bed rest, and exercise training. Male Sprague-Dawley rats (n = 48) were assigned to sham ambulatory, sham hindlimb unloading, burn ambulatory, or burn plus hindlimb unloading groups. Rats received a 40% TBSA scald burn or sham treatments and were ambulatory or hindlimb unloaded. Rats were further assigned to exercise or no exercise. Plantaris tissues were harvested on day 14 and pooled to analyze for microRNA and gene expression profiles. Compared with the sham ambulatory-no exercise group, 73, 79, and 80 microRNAs were altered 2-fold in the burn ambulatory, sham hindlimb unloading, and burn hindlimb unloading groups, all with no exercise, respectively. More than 70% of microRNAs were upregulated in response to burn and hindlimb unloading, whereas 60% microRNA of the profile decreased in hindlimb unloaded burn rats with exercise training. MiR-182 was the most affected in rat muscle. Gene ontology biological process and pathway analysis showed that the oxidative stress pathway was most stimulated in the hindlimb unloaded burn rats; while in response to exercise training, all genes in related pathways such as hypermetabolic, inflammation, and blood coagulation were alleviated. MicroRNAs and transcript gene profiles were altered in burn and hindlimb unloading groups, with additive effects on hindlimb unloaded burn rats. The altered genes' signal pathways were associated with muscle mass loss and function impairment. Muscle improvement with exercise training was observed in gene levels with microRNA alterations as well.

Characterization and differential expression of microRNA in skeletal muscle of Laiwu and Yorkshire pig breeds

The domestic pig is an important agricultural animal used as a source of meat worldwide. As an important resource, Laiwu pig has some special characteristics compared with Yorkshire pig. It is necessary to identify the differentially expressed microRNAs (miRNAs) in skeletal muscle between two pig breeds. Therefore, we herein performed a comprehensive investigation for miRNA transcriptome of skeletal muscle from the two pig breeds. On average, we obtained approximately 13,493,607 clean reads in Laiwu pig and 12,938,257 clean reads in Yorkshire pig in this study. Totally, 265 known miRNAs were identified, and among these, 229 miRNAs were expressed in all the six pigs. From the 265 known miRNAs, 19 of these miRNAs were found significantly differentially expressed (q < 0.05). Of these significantly expressed miRNAs, seven miRNAs were significantly up-regulated and 12 miRNAs were down-regulated in Laiwu pig compared with Yorkshire pig. Among the significantly expressed miRNAs, ssc-miR-194-5p has the largest fold-change (6.35-fold). Ten differentially expressed miRNAs selected from high throughput sequencing were confirmed by real-time RT-PCR (RT-qPCR), and the results indicated that the expression patterns were consistent with sequencing results. Our results not only present a comprehensive miRNA transcriptome profile of skeletal muscle between two pig breeds with distinct phenotypes, but also could be useful for investigating the functions of differentially expressed miRNAs associated with the phenotype traits.

Skeletal Muscle MicroRNAs 206 and 424 are Prognostic Markers in Amyotrophic Lateral Sclerosis (ALS) (P5.042)

Skeletal Muscle MicroRNAs 206 and 424 are Prognostic Markers in Amyotrophic Lateral Sclerosis (ALS) (P5.042)

Selective Modulation of MicroRNA Expression with Protein Ingestion Following Concurrent Resistance and Endurance Exercise in Human Skeletal Muscle.

We examined changes in the expression of 13 selected skeletal muscle microRNAs (miRNAs) implicated in exercise adaptation responses following a single bout of concurrent exercise. In a randomized cross-over design, seven healthy males undertook a single trial consisting of resistance exercise (8 × 5 leg extension, 80% 1 Repetition Maximum) followed by cycling (30 min at ~70% VO2peak) with either post-exercise protein (PRO: 25 g whey protein) or placebo (PLA) ingestion. Muscle biopsies (vastus lateralis) were obtained at rest and 4 h post-exercise. Detection of miRNA via quantitative Polymerase Chain Reaction (qPCR) revealed post-exercise increases in miR-23a-3p (~90%), miR-23b-3p (~39%), miR-133b (~80%), miR-181-5p (~50%), and miR-378-5p (~41%) at 4 h post-exercise with PRO that also resulted in higher abundance compared to PLA (P < 0.05). There was a post-exercise decrease in miR-494-3p abundance in PLA only (~88%, P < 0.05). There were no changes in the total abundance of target proteins post-exercise or between conditions. Protein ingestion following concurrent exercise can modulate the expression of miRNAs implicated in exercise adaptations compared to placebo. The selective modulation of miRNAs with target proteins that may prioritize myogenic compared to oxidative/metabolic adaptive responses indicate that miRNAs can play a regulatory role in the molecular machinery enhancing muscle protein synthesis responses with protein ingestion following concurrent exercise.

MicroRNAs In Skeletal Muscle and Their Response to Resistance Exercise in Insulin Resistant Diabetes

microRNAs In Skeletal Muscle and Their Response to Resistance Exercise in Insulin Resistant Diabetes

Glucose tolerance is associated with differential expression of microRNAs in skeletal muscle: results from studies of twins with and without type 2 diabetes.

Aims/hypothesisWe aimed to identify microRNAs (miRNAs) associated with type 2 diabetes and risk of developing the disease in skeletal muscle biopsies from phenotypically well-characterised twins.MethodsWe measured muscle miRNA levels in monozygotic (MZ) twins discordant for type 2 diabetes using arrays. Further investigations of selected miRNAs included target prediction, pathway analysis, silencing in cells and association analyses in a separate cohort of 164 non-diabetic MZ and dizygotic twins. The effects of elevated glucose and insulin levels on miRNA expression were examined, and the effect of low birthweight (LBW) was studied in rats.ResultsWe identified 20 miRNAs that were downregulated in MZ twins with diabetes compared with their non-diabetic co-twins. Differences for members of the miR-15 family (miR-15b and miR-16) were the most statistically significant, and these miRNAs were predicted to influence insulin signalling. Indeed, miR-15b and miR-16 levels were associated with levels of key insulin signalling proteins, miR-15b was associated with the insulin receptor in non-diabetic twins and knockdown of miR-15b/miR-16 in myocytes changed the levels of insulin signalling proteins. LBW in twins and undernutrition during pregnancy in rats were, in contrast to overt type 2 diabetes, associated with increased expression of miR-15b and/or miR-16. Elevated glucose and insulin suppressed miR-16 expression in vitro.ConclusionsType 2 diabetes is associated with non-genetic downregulation of several miRNAs in skeletal muscle including miR-15b and miR-16, potentially targeting insulin signalling. The paradoxical findings in twins with overt diabetes and twins at increased risk of the disease underscore the complexity of the regulation of muscle insulin signalling in glucose homeostasis.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-014-3434-2) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Periconceptional undernutrition programs changes in insulin-signaling molecules and microRNAs in skeletal muscle in singleton and twin fetal sheep.

Maternal undernutrition around the time of conception is associated with an increased risk of insulin resistance in adulthood. We determined the effect of maternal undernutrition in the periconceptional period (PCUN, i.e., 60 days prior to 6 days after conception) and the preimplantation period (PIUN, i.e., 0-6 days after conception) on mRNA expression and protein abundance of key insulin-signaling molecules as well as the global microRNA expression in quadriceps muscle of singleton and twin fetal sheep in late gestation. In singleton fetuses, exposure to PCUN resulted in lower protein abundance of PIK3CB (P < 0.01), PRKCZ (P < 0.05), and pPRKCZ (Thr410) (P < 0.05) in skeletal muscle compared to controls. In PIUN singletons, there was a higher protein abundance of IRS1 (P < 0.05), PDPK1 (P < 0.05), and SLC2A4 (P < 0.05) compared to controls. In twins, PCUN resulted in higher protein abundance of IRS1 (P < 0.05), AKT2 (P < 0.05), PDPK1 (P < 0.05), and PRKCZ (P < 0.001), while PIUN also resulted in higher protein abundance of IRS1 (P < 0.05), PRKCZ (P < 0.001), and SLC2A4 (P < 0.05) in fetal muscle compared to controls. There were specific patterns of the types and direction of changes in the expression of 22 microRNAs in skeletal muscle after exposure to PCUN or PIUN and clear differences in these patterns between singleton and twin pregnancies. These findings provide evidence that maternal undernutrition around the time of conception induces changes in the expression of microRNAs, which may play a role in altering the abundance of the key insulin-signaling molecules in skeletal muscle and in the association between PCUN undernutrition and insulin resistance in adult life.

Measuring microRNA reporter activity in skeletal muscle using hydrodynamic limb vein injection of plasmid DNA combined with in vivo imaging

BackgroundmicroRNA regulation plays an important role in the remodeling that occurs in response to pathologic and physiologic stimuli in skeletal muscle. In response to stress, microRNAs are dynamically regulated, resulting in a widespread “fine-tuning” of gene expression. An understanding of this dynamic regulation is critical to targeting future therapeutic strategies. Experiments elucidating this dynamic regulation have typically relied on in vitro reporter assays, ex vivo sample analysis, and transgenic mouse studies. Surprisingly, no experimental method to date allows rapid in vivo analysis of microRNA activity in mammals.MethodsTo improve microRNA studies we have developed a novel reporter assay for the measurement of skeletal muscle microRNA activity in vivo. To minimize muscle damage, hydrodynamic limb vein injection was used for the introduction of plasmid DNA encoding bioluminescent and fluorescent reporters, including click-beetle luciferase and the far-red fluorescent protein mKATE. We then applied this technique to the measurement of miR-206 activity in dystrophic mdx4cv animals.ResultsWe found that hydrodynamic limb vein injection is minimally damaging to myofibers, and as a result no induction of muscle-specific miR-206 (indicative of an injury response) was detected. Unlike intramuscular injection or electroporation, we found that hydrodynamic limb vein injection results in dispersed reporter expression across multiple hindlimb muscle groups. Additionally, by utilizing click-beetle luciferase from Pyrophorus plagiophthalamus as a reporter and the far-red fluorescent protein mKATE for normalization, we show as a proof of principle that we can detect elevated miR-206 activity in mdx4cv animals when compared to C57Bl/6 controls.ConclusionHydrodynamic limb vein injection of plasmid DNA followed by in vivo bioluminescent imaging is a novel assay for the detection of reporter activity in skeletal muscle in vivo. We believe that this method will allow for the rapid and precise detection of both transcriptional and post-transcriptional regulation of gene expression in response to skeletal muscle stress. Additionally, given the post-mitotic status of myofibers and stable expression of plasmid DNA, we believe this method will reduce biological variability in animal studies by allowing longitudinal studies of the same animal cohort.

MicroRNAs in skeletal muscle and their regulation with exercise, ageing, and disease

Skeletal muscle makes up approximately 40% of the total body mass, providing structural support and enabling the body to maintain posture, to control motor movements and to store energy. It therefore plays a vital role in whole body metabolism. Skeletal muscle displays remarkable plasticity and is able to alter its size, structure and function in response to various stimuli; an essential quality for healthy living across the lifespan. Exercise is an important stimulator of extracellular and intracellular stress signals that promote positive adaptations in skeletal muscle. These adaptations are controlled by changes in gene transcription and protein translation, with many of these molecules identified as potential therapeutic targets to pharmacologically improve muscle quality in patient groups too ill to exercise. MicroRNAs (miRNAs) are recently identified regulators of numerous gene networks and pathways and mainly exert their effect by binding to their target messenger RNAs (mRNAs), resulting in mRNA degradation or preventing protein translation. The role of exercise as a regulatory stimulus of skeletal muscle miRNAs is now starting to be investigated. This review highlights our current understanding of the regulation of skeletal muscle miRNAs with exercise and disease as well as how they may control skeletal muscle health.

MicroRNAs in skeletal muscle: their role and regulation in development, disease and function

Maintaining skeletal muscle function throughout the lifespan is a prerequisite for good health and independent living. For skeletal muscle to consistently function at optimal levels, the efficient activation of processes that regulate muscle development, growth, regeneration and metabolism is required. Numerous conditions including neuromuscular disorders, physical inactivity, chronic disease and ageing are associated with perturbations in skeletal muscle function. A loss or reduction in skeletal muscle function often leads to increased morbidity and mortality either directly, or indirectly, via the development of secondary diseases such as diabetes, obesity, cardiovascular and respiratory disease. Identifying mechanisms which influence the processes regulating skeletal muscle function is a key priority. The discovery of microRNAs (miRNAs) provides a new avenue that will extend our knowledge of factors controlling skeletal muscle function. miRNAs may also improve our understanding and application of current therapeutic approaches as well as enable the identification of new therapeutic strategies and targets aimed at maintaining and/or improving skeletal muscle health. This review brings together the latest developments in skeletal muscle miRNA biology and focuses on their role and regulation under physiological and patho-physiological conditions with an emphasis on: myogenesis, hypertrophy, atrophy and regeneration; exercise and nutrition; muscle disease, ageing, diabetes and obesity.

High responders to resistance exercise training demonstrate differential regulation of skeletal muscle microRNA expression

MicroRNAs (miRNA), small noncoding RNA molecules, may regulate protein synthesis, while resistance exercise training (RT) is an efficient strategy for stimulating muscle protein synthesis in vivo. However, RT increases muscle mass, with a very wide range of effectiveness in humans. We therefore determined the expression level of 21 abundant miRNAs to determine whether variation in these miRNAs was able to explain the variation in RT-induced gains in muscle mass. Vastus lateralis biopsies were obtained from the top and bottom ∼20% of responders from 56 young men who undertook a 5 day/wk RT program for 12 wk. Training-induced muscle mass gain was determined by dual-energy X-ray absorptiometry, and fiber size was evaluated by histochemistry. The expression level of each miRNA was quantified using TaqMan-based quantitative PCR, with the analysis carried out in a blinded manner. Gene ontology and target gene profiling were used to predict the potential biological implications. Of the 21 mature miRNAs examined, 17 were stable during RT in both groups. However, miR-378, miR-29a, miR-26a, and miR-451 were differentially expressed between low and high responders. miR-378, miR-29a, and miR-26a were downregulated in low responders and unchanged in high responders, while miR-451 was upregulated only in low responders. Interestingly, the training-induced change in miR-378 abundance was positively correlated with muscle mass gains in vivo. Gene ontology analysis of the target gene list of miR-378, miR-29a, miR-26a, and miR-451, from the weighted cumulative context ranking methodology, indicated that miRNA changes in the low responders may be compensatory, reflecting a failure to "activate" growth and remodeling genes. We report, for the first time, that RT-induced hypertrophy in human skeletal muscle is associated with selected changes in miRNA abundance. Our analysis indicates that miRNAs may play a role in the phenotypic change and pronounced intergroup variation in the RT response.

Aging differentially affects human skeletal muscle microRNA expression at rest and after an anabolic stimulus of resistance exercise and essential amino acids

Sarcopenia, skeletal muscle loss during aging, is associated with increased falls, fractures, morbidity, and loss of independence. MicroRNAs (miRNAs) are novel posttranscriptional regulators. The role of miRNAs in cell size regulation after an anabolic stimulus in human skeletal muscle is unknown. We hypothesized that aging would be associated with a differential expression of skeletal muscle primary miRNA (pri-miRNA) and mature miRNA (miR). To test this hypothesis, we used real-time PCR and immunoblotting before and after an anabolic stimulus (resistance exercise + ingestion of a 20-g leucine-enriched essential amino acid solution) to measure the expression of muscle-specific miRNAs (miR-1, miR-133a, and miR-206), upstream regulators (MyoD and myogenin), and downstream targets [insulin-like growth factor-I, histone deacetylase-4, myocyte enhancing factor-2, and Ras homolog enriched in brain (Rheb)] in skeletal muscle of young and older men. Muscle biopsies were obtained at baseline and 3 and 6 h after exercise. At baseline, we found pri-miRNA-1-1, -1-2, -133a-1, and -133a-2 expression elevated in older compared with young men (P < 0.05). Pri-miRNA-1-2, -133a-1, and -133a-2 were reduced at 6 h after exercise only in the young men compared with baseline, whereas pri-miRNA-206 was elevated at different postexercise time points in older and young men (P < 0.05). Compared with baseline, miR-1 was reduced only in the young men, whereas Rheb protein was increased in both age groups after the anabolic stimulus (P < 0.05). We conclude that skeletal muscle primary and mature miRNA expression in young men is readily altered by an anabolic stimulus of resistance exercise + essential amino acid ingestion. However, aging is associated with higher basal skeletal muscle primary miRNA expression and a dysregulated miRNA response after the anabolic stimulus.

MicroRNA-206 is overexpressed in the diaphragm but not the hindlimb muscle of mdx mouse

MicroRNAs are highly conserved, noncoding RNAs involved in posttranscriptional gene silencing. MicroRNAs have been shown to be involved in a range of biological processes, including myogenesis and muscle regeneration. The objective of this study was to test the hypothesis that microRNA expression is altered in dystrophic muscle, with the greatest change occurring, of the muscles examined, in the diaphragm. The expression of the muscle-enriched microRNAs was determined in the soleus, plantaris, and diaphragm muscles of control and dystrophin-deficient (mdx) mice by semiquantitative PCR. In the soleus and plantaris, expression of the mature microRNA 133a (miR-133a) and miR-206, respectively, was decreased by approximately 25%, whereas in the diaphragm, miR-206 expression increased by 4.5-fold relative to control. The increased expression of miR-206 in the mdx diaphragm was paralleled by a 4.4-fold increase in primary miRNA-206 (pri-miRNA-206) transcript level. Expression of Myod1 was elevated 2.7-fold only in the mdx diaphragm, consistent with an earlier finding demonstrating Myod1 can activate pri-miRNA-206 transcription. Transcript levels of Drosha and Dicer, major components of microRNA biogenesis pathway, were unchanged in mdx muscle, suggesting the pathway is not altered under dystrophic conditions. Previous in vitro analysis found miR-206 was capable of repressing utrophin expression; however, under dystrophic conditions, both utrophin transcript and protein levels were significantly increased by 69% and 3.9-fold, respectively, a finding inconsistent with microRNA regulation. These results are the first to report alterations in expression of muscle-enriched microRNAs in skeletal muscle of the mdx mouse, suggesting microRNAs may have a role in the pathophysiology of muscular dystrophy.