Human primary myoblasts derived from paraspinal muscle reflect donor age as an experimental model of sarcopenia.

BACKGROUND: Low back pain is a general phenomenon of aging, and surgery is an unavoidable choice to relieve severe back pain. The discarded surgical site during surgery is of high value for muscle and muscle-related research. This study investigated the age-dependent properties of patients' paraspinal muscles at the cellular level. METHODS: To define an association of paraspinal muscle degeneration with sarcopenia, we analyzed lumbar paraspinal muscle and myoblasts isolated from donors of various ages (25-77 years). Preoperative evaluations were performed by bioimpedance analysis using the InBody 720, magnetic resonance (MR) imaging of the lumbar spine, and lumbar extension strength using a lumbar extension dynamometer. In addition, the growth and differentiation capacity of myoblasts obtained from the donor was determined using proliferation assay and western blotting. RESULTS: The cross-sectional area of the lumbar paraspinal muscle decreased with age and was also correlated with the appendicular skeletal muscle index (ASM/height2). Human primary myoblasts isolated from paraspinal muscle preserved their proliferative capacity in vitro, which tended to decrease with donor age. The age-dependent decline in myoblast proliferation was correlated with levels of cell cycle inhibitory proteins (p16INK4a, p21CIP1, and p27KIP1) associated with cellular senescence. Primary myoblasts isolated from younger donors differentiated into multinucleate myotubes earlier and at a higher rate than those from older donors in vitro. Age-dependent decline in myogenic potential of the isolated primary myoblasts was likely correlated with the inactivation of myogenic transcription factors such as MyoD, myogenin, and MEF2c. CONCLUSIONS: Myoblasts isolated from human paraspinal muscle preserve myogenic potential that correlates with donor age, providing an in vitro model of sarcopenia.

The role of non-coding RNAs in muscle aging: regulatory mechanisms and therapeutic potential.

Muscle aging is a complex physiological process that leads to the progressive decline in muscle mass and function, contributing to debilitating conditions in the elderly such as sarcopenia. In recent years, non-coding RNAs (ncRNAs) have been increasingly recognized as major regulators of muscle aging and related cellular processes. Here, we comprehensively review the emerging role of ncRNAs, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), in the regulation of muscle aging. We also discuss how targeting these ncRNAs can be explored for the development of novel interventions to combat age-related muscle decline. The insights provided in this review offer a promising avenue for future research and therapeutic strategies aimed at improving muscle health during aging.

Genome-wide analysis of a cellular exercise model based on electrical pulse stimulation.

Skeletal muscle communicates with other organs via myokines, which are secreted by muscle during exercise and exert various effects. Despite much investigation of the exercise, the underlying molecular mechanisms are still not fully understood. Here, we applied an in vitro exercise model in which cultured C2C12 myotubes were subjected to electrical pulse stimulation (EPS), which mimics contracting muscle. Based on the significantly up- and down-regulated genes in EPS, we constructed an in silico model to predict exercise responses at the transcriptional level. The in silico model revealed similarities in the transcriptomes of the EPS and exercised animals. Comparative analysis of the EPS data and exercised mouse muscle identified putative biomarkers in exercise signaling pathways and enabled to discover novel exercise-induced myokines. Biochemical analysis of selected exercise signature genes in muscle from exercised mice showed that EPS mimics in vivo exercise, at least in part, at the transcriptional level. Consequently, we provide a novel myokine, Amphiregulin (AREG), up-regulated both in vitro and in vivo, that would be a potential target for exercise mimetics.

A subset of microRNAs in the Dlk1-Dio3 cluster regulates age-associated muscle atrophy by targeting Atrogin-1.

BACKGROUND: The microRNAs (miRNAs) down-regulated in aged mouse skeletal muscle were mainly clustered within the delta-like homologue 1 and the type III iodothyronine deiodinase (Dlk1-Dio3) genomic region. Although clustered miRNAs are coexpressed and regulate multiple targets in a specific signalling pathway, the function of miRNAs in the Dlk1-Dio3 cluster in muscle aging is largely unknown. We aimed to ascertain whether these miRNAs play a common role to regulate age-related muscle atrophy. METHODS: To examine anti-atrophic effect of miRNAs, we individually transfected 42 miRNA mimics in fully differentiated myotubes and analysed their diameters. The luciferase reporter assay using target 3' untranslated region (UTR) and RNA pull-down assay were employed to ascertain the target predicted by the TargetScan algorithm. To investigate the therapeutic potential of the miRNAs in vivo, we generated adeno-associated virus (AAV) serotype 9 expressing green fluorescent protein (GFP) (AAV9-GFP) bearing miR-376c-3p and infected it into the tibialis anterior muscle of old mice. We performed morphometric analysis and measured ex vivo isometric force using a force transducer. Human gluteus maximus muscle tissues (ages ranging from 25 to 80 years) were used to investigate expression levels of the conserved miRNAs in the Dlk1-Dio3 cluster. RESULTS: We found that the majority of miRNAs (33 out of 42 tested) in the cluster induced anti-atrophic phenotypes in fully differentiated myotubes with increasing their diameters. Eighteen of these miRNAs, eight of which are conserved in humans, harboured predicted binding sites in the 3' UTR of muscle atrophy gene-1 (Atrogin-1) encoding a muscle-specific E3 ligase. Direct interactions were identified between these miRNAs and the 3' UTR of Atrogin-1, leading to repression of Atrogin-1 and thereby induction of eIF3f protein content, in both human and mouse skeletal muscle cells. Intramuscular delivery of AAV9 expressing miR-376c-3p, one of the most effective miRNAs in myotube thickening, dramatically ameliorated skeletal muscle atrophy and improved muscle function, including isometric force, twitch force, and fatigue resistance in old mice. Consistent with our findings in mice, the expression of miRNAs in the cluster was significantly down-regulated in human muscle from individuals > 50 years old. CONCLUSIONS: Our study suggests that genetic intervention using a muscle-directed miRNA delivery system has therapeutic efficacy in preventing Atrogin-1-mediated muscle atrophy in sarcopenia.

Plasma proteomic profiling of young and old mice reveals cadherin-13 prevents age-related bone loss.

The blood exhibits a dynamic flux of proteins that are secreted by the tissues and cells of the body. To identify novel aging-related circulating proteins, we compared the plasma proteomic profiles of young and old mice using tandem mass spectrometry. The expression of 134 proteins differed between young and old mice. We selected seven proteins that were expressed at higher levels in young mice, and confirmed their plasma expression in immunoassays. The plasma levels of anthrax toxin receptor 2 (ANTXR2), cadherin-13 (CDH-13), scavenger receptor cysteine-rich type 1 protein M130 (CD163), cartilage oligomeric matrix protein (COMP), Dickkopf-related protein 3 (DKK3), periostin, and secretogranin-1 were all confirmed to decrease with age. We then investigated whether any of the secreted proteins influenced bone metabolism and found that CDH-13 inhibited osteoclast differentiation. CDH 13 treatment suppressed the receptor activator of NF-κB ligand (RANKL) signaling pathway in bone marrow-derived macrophages, and intraperitoneal administration of CDH-13 delayed age-related bone loss in the femurs of aged mice. These findings suggest that low plasma CDH-13 expression in aged mice promotes aging-associated osteopenia by facilitating excessive osteoclast formation. Thus, CDH-13 could have therapeutic potential as a protein drug for the prevention of osteopenia.

miR-3074-3p promotes myoblast differentiation by targeting Cav1.

Muscle fibers are generally formed as multinucleated fibers that are differentiated from myoblasts. Several reports have identified transcription factors and proteins involved in the process of muscle differentiation, but the roles of microRNAs (miRNAs) in myogenesis remain unclear. Here, comparative analysis of the miRNA expression profiles in mouse myoblasts and gastrocnemius (GA) muscle uncovered miR-3074-3p as a novel miRNA showing markedly reduced expression in fully differentiated adult skeletal muscle. Interestingly, elevating miR-3074-3p promoted myogenesis in C2C12 cells, primary myoblasts, and HSMMs, resulting in increased mRNA expression of myogenic makers such as Myog and MyHC. Using a target prediction program, we identified Caveolin-1 (Cav1) as a target mRNA of miR-3074-3p and verified that miR-3074-3p directly interacts with the 3' untranslated region (UTR) of Cav1 mRNA. Consistent with the findings in miR-3074-3p-overexpressing myoblasts, knockdown of Cav1 promoted myogenesis in C2C12 cells and HSMMs. Taken together, our results suggest that miR-3074-3p acts a positive regulator of myogenic differentiation by targeting Cav1. [BMB Reports 2020; 53(5): 278-283].

Comprehensive miRNA Profiling of Skeletal Muscle and Serum in Induced and Normal Mouse Muscle Atrophy During Aging.

Age-associated loss of muscle mass and function is a major cause of morbidity and mortality in the elderly adults. Muscular atrophy can also be induced by disuse associated with long-term bed rest or disease. Although miRNAs regulate muscle growth, regeneration, and aging, their potential role in acute muscle atrophy is poorly understood. Furthermore, alterations in circulating miRNA levels have been shown to occur during aging but their potential as noninvasive biomarkers for muscle atrophy remains largely unexplored. Here, we report comprehensive miRNA expression profiles by miRNA-seq analysis in tibialis anterior muscle and serum of a disuse-induced atrophy mouse model, mimicking the acute atrophy following long-term bed rest, as compared to those of young and old mice. Comparative analysis and validation studies have revealed that miR-455-3p was significantly decreased in muscle of both induced-atrophy model and old mice, whereas miR-434-3p was decreased in both serum and muscle of old mice, as compared to young mice. Furthermore, upregulation of miR-455-3p in fully differentiated C2C12 myoblasts induced a hypertrophic phenotype. These results suggest that deregulation of miR-455-3p may play a functional role in muscle atrophy and miR-434-3p could be a candidate serum biomarker of muscle aging.

microRNA for determining the age-related myogenic capabilities of skeletal muscle.

Skeletal muscle exhibits a loss of muscle mass and function with age. Decreased regenerative potential of muscle stem/progenitor cells is a major underlying cause of sarcopenia. We analyzed microRNAs (miRNA) that are differentially expressed in young and old myoblasts, to identify novel intrinsic factors that play a degenerative role in aged skeletal muscle. miR-431, one of decreasing miRNAs in old myoblasts, improved the myogenic differentiation when overexpressed in old myoblast, but suppressed their myogenic capability in knockdowned young myoblasts. We found that miR-431 directly binds to 3`untranslated regions (UTR) of Smad4 mRNA, and decreases its expression. Given that SMAD4 is one of the downstream effectors of TGF-ß, a well-known degenerative signaling pathway in myogenesis, the decreased miR-431 in old myoblast causes SMAD4 elevation, thus resulting in defective myogenesis. Exogenous expression of miR-431 greatly improved the muscle regeneration in the cardiotoxin-injured hindlimb muscle of old mice by reducing SMAD4 levels. Since the miR-431 seed sequence is conserved in human SMAD4 3'UTR, miR-431 regulates the myogenic capacity of human skeletal myoblasts in the same manner. Our results suggest that age-associated miR-431 is required for the maintenance of the myogenic capability in myoblasts, thus underscoring its potential as a therapeutic target to slow down muscle aging.

miR-431 promotes differentiation and regeneration of old skeletal muscle by targeting Smad4.

The myogenic capacity of myoblasts decreases in skeletal muscle with age. In addition to environmental factors, intrinsic factors are important for maintaining the regenerative potential of muscle progenitor cells, but their identities are largely unknown. Here, comparative analysis of microRNA (miRNA) expression profiles in young and old myoblasts uncovered miR-431 as a novel miRNA showing markedly reduced abundance in aged myoblasts. Importantly, elevating miR-431 improved the myogenic capacity of old myoblasts, while inhibiting endogenous miR-431 lowered myogenesis. Bioinformatic and biochemical analyses revealed that miR-431 directly interacted with the 3' untranslated region (UTR) of Smad4 mRNA, which encodes one of the downstream effectors of TGF-ß signaling. In keeping with the low levels of miR-431 in old myoblasts, SMAD4 levels increased in this myoblast population. Interestingly, in an in vivo model of muscle regeneration following cardiotoxin injury, ectopic miR-431 injection greatly improved muscle regeneration and reduced SMAD4 levels. Consistent with the finding that the mouse miR-431 seed sequence in the Smad4 3' UTR is conserved in the human SMAD4 3' UTR, inhibition of miR-431 also repressed the myogenic capacity of human skeletal myoblasts. Taken together, our results suggest that the age-associated miR-431 plays a key role in maintaining the myogenic ability of skeletal muscle with age.

Peroxiredoxin 3 has a crucial role in the contractile function of skeletal muscle by regulating mitochondrial homeostasis.

Antioxidant systems against reactive oxygen species (ROS) are important factors in regulating homeostasis in various cells, tissues, and organs. Although ROS are known to cause to muscular disorders, the effects of mitochondrial ROS in muscle physiology have not been fully understood. Here, we investigated the effects of ROS on muscle mass and function using mice deficient in peroxiredoxin 3 (Prx3), which is a mitochondrial antioxidant protein. Ablation of Prx3 deregulated the mitochondrial network and membrane potential of myotubes, in which ROS levels were increased. We showed that the DNA content of mitochondria and ATP production were also reduced in Prx3-KO muscle. Of note, the mitofusin 1 and 2 protein levels decreased in Prx3-KO muscle, a biochemical evidence of impaired mitochondrial fusion. Contractile dysfunction was examined by measuring isometric forces of isolated extensor digitorum longus (EDL) and soleus muscles. Maximum absolute forces in both the EDL and the soleus muscles were not significantly affected in Prx3-KO mice. However, fatigue trials revealed that the decrease in relative force was greater and more rapid in soleus from Prx3-KO compared to wild-type mice. Taken together, these results suggest that Prx3 plays a crucial role in mitochondrial homeostasis and thereby controls the contractile functions of skeletal muscle.