Role of oxidation of excitation-contraction coupling machinery in age-dependent loss of muscle function in C. elegans

ABSTRACTAge-dependent loss of body wall muscle function and impaired locomotion occur within 2 weeks in C. elegans; however, the underlying mechanism has not been fully elucidated. In humans, age-dependent loss of muscle function occurs at about 80 years of age and has been linked to dysfunction of ryanodine receptor (RyR)/intracellular calcium (Ca2+) release channels on the sarcoplasmic reticulum (SR). Mammalian skeletal muscle RyR1 channels undergo age-related remodeling due to oxidative overload, leading to loss of the stabilizing subunit calstabin1 (FKBP12) from the channel macromolecular complex. This destabilizes the closed state of the channel resulting in intracellular Ca2+ leak, reduced muscle function, and impaired exercise capacity. We now show that the C. elegans RyR homolog, UNC-68, exhibits a remarkable degree of evolutionary conservation with mammalian RyR channels and similar age-dependent dysfunction. Like RyR1 in mammals UNC-68 encodes a protein that comprises a macromolecular complex which includes the calstabin1 homolog FKB-2 and is immunoreactive with antibodies raised against the RyR1 complex. Further, as in aged mammals, UNC-68 is oxidized and depleted of FKB-2 in an age-dependent manner, resulting in “leaky” channels, depleted SR Ca2+ stores, reduced body wall muscle Ca2+ transients, and age-dependent muscle weakness. FKB-2 (ok3007)-deficient worms exhibit reduced exercise capacity. Pharmacologically induced oxidization of UNC-68 and depletion of FKB-2 from the channel independently caused reduced body wall muscle Ca2+ transients. Preventing FKB-2 depletion from the UNC-68 macromolecular complex using the Rycal drug S107 improved muscle Ca2+ transients and function. Taken together, these data suggest that UNC-68 oxidation plays a role in age-dependent loss of muscle function. Remarkably, this age-dependent loss of muscle function induced by oxidative overload, which takes ~2 years in mice and ~80 years in humans, occurs in less than 2-3 weeks in C. elegans, suggesting that reduced anti-oxidant capacity may contribute to the differences in life span amongst species.

Transcriptome-wide N6-Methyladenosine Methylome Profiling Reveals m6A Regulation of Skeletal Myoblast Differentiation in Cattle (Bos taurus)

N6-methyladenosine (m6A) is the most prevalent methylation modification of eukaryotic mRNA, and it plays an important role in regulating gene expression. Previous studies have found that m6A methylation plays a role in mammalian skeletal muscle development. However, the effect of m6A on bovine skeletal myogenesis are still unclear. Here, we selected proliferating myoblasts (GM) and differentiated myotubes (on the 4th day of differentiation, DM) for m6A-seq and RNA-seq to explore the m6A methylation modification pattern during bovine skeletal myogenesis. m6A-seq analysis revealed that m6A methylation was an abundant modification of the mRNA in bovine myoblasts and myotubes. We scanned 5,691–8,094 m6A-modified transcripts, including 1,437 differentially methylated genes (DMGs). GO and KEGG analyses revealed that DMGs were primarily involved in transcriptional regulation and RNA metabolism, as well as insulin resistance and metabolic pathways related to muscle development. The combined analysis further identified 268 genes that had significant changes at both m6A and mRNA levels, suggesting that m6A modification may regulate myoblast differentiation by mediating the expression of these genes. Furthermore, we experimentally confirmed four genes related to myogenesis, including MYOZ2, TWIST1, KLF5 and MYOD1, with differential changes in both m6A and mRNA levels during bovine myoblast differentiation, indicating that they can be potential candidate targets for m6A regulation of skeletal myogenesis. Our results may provide new insight into molecular genetics and breeding of beef cattle, and provide a reference for investigating the mechanism of m6A regulating skeletal muscle development.

A Quantitative Proteomics Approach to Gain Insight into NRF2-KEAP1 Skeletal Muscle System and Its Cysteine Redox Regulation

Mammalian skeletal muscle (SkM) tissue engages the Nrf2-Keap1-dependent antioxidant defense mechanism to respond adaptively to stress. Redox homeostasis mediated by the reversible modification of selective cysteines is the prevalent mode of regulation. The protein targets of SkM redox regulation are largely unknown. We previously reported the proteomic profiles of soleus (Sol) and extensor digitorum longus (EDL) with Nrf2 or Keap1 gene deletion, using SkM-specific Nrf2 or Keap1 knockout models; iMS-Nrf2flox/flox; and iMS-Keap1flox/flox. Here, we employed these two animal models to understand the global expression profile of red tibialis anterior (RTA) using a label free approach and its redox proteomics using iodoacetyl tandem mass tag (iodoTMTTM)-labeled cysteine quantitation. We quantified 298 proteins that were significantly altered globally in the RTA with Nrf2 deficiency but only 21 proteins in the Keap1 KO samples. These proteins are involved in four intracellular signaling pathways: sirtuin signaling, Nrf2 mediated oxidative stress response, oxidative phosphorylation, and mitochondrion dysfunction. Moreover, we identified and quantified the cysteine redox peptides of 34 proteins, which are associated with mitochondrial oxidative phosphorylation, energy metabolism, and extracellular matrix. Our findings suggest that Nrf2-deficient RTA is implicated in metabolic myopathy, mitochondrial disorders, and motor dysfunction, possibly due to an enhanced oxidative modification of the structure and functional proteins in skeletal myocytes.

Characterization of Long Non-coding RNAs Modified by m6A RNA Methylation in Skeletal Myogenesis

Proper development of mammalian skeletal muscle relies on precise gene expression regulation. Our previous studies revealed that muscle development is regulated by both mRNA and long non-coding RNAs (lncRNAs). Accumulating evidence has demonstrated that N6-methyladenosine (m6A) plays important roles in various biological processes, making it essential to profile m6A modification on a transcriptome-wide scale in developing muscle. Patterns of m6A methylation in lncRNAs in developing muscle have not been uncovered. Here, we reveal differentially expressed lncRNAs and report temporal m6A methylation patterns in lncRNAs expressed in mouse myoblasts and myotubes by RNA-seq and methylated RNA immunoprecipitation (MeRIP) sequencing. Many lncRNAs exhibit temporal differential expression, and m6A-lncRNAs harbor the consensus m6A motif “DRACH” along lncRNA transcripts. Interestingly, we found that m6A methylation levels of lncRNAs are positively correlated with the transcript abundance of lncRNAs. Overexpression or knockdown of m6A methyltransferase METTL3 alters the expression levels of these lncRNAs. Furthermore, we highlight that the function of m6A genic lncRNAs might correlate to their nearby mRNAs. Our work reveals a fundamental expression reference of m6A-mediated epitranscriptomic modifications in lncRNAs that are temporally expressed in developing muscle.

Male-Biased gga-miR-2954 Regulates Myoblast Proliferation and Differentiation of Chicken Embryos by Targeting YY1

Previous studies have shown that gga-miR-2954 was highly expressed in the gonads and other tissues of male chickens, including muscle tissue. Yin Yang1 (YY1), which has functions in mammalian skeletal muscle development, was predicted to be a target gene of gga-miR-2954. The purpose of this study was to investigate whether gga-miR-2954 plays a role in skeletal muscle development by targeting YY1, and evaluate its function in the sexual dimorphism development of chicken muscle. Here, all the temporal and spatial expression profiles in chicken embryonic muscles showed that gga-miR-2954 is highly expressed in males and mainly localized in cytoplasm. Gga-miR-2954 exhibited upregulated expression of in vitro myoblast differentiation stages. Next, through the overexpression and loss-of-function experiments performed in chicken primary myoblasts, we found that gga-miR-2954 inhibited myoblast proliferation but promoted differentiation. During myogenesis, gga-miR-2954 could suppress the expression of YY1, which promoted myoblast proliferation and inhibited the process of myoblast cell differentiation into multinucleated myotubes. Overall, these findings reveal a novel role of gga-miR-2954 in skeletal muscle development through its function of the myoblast proliferation and differentiation by suppressing the expression of YY1. Moreover, gga-miR-2954 may contribute to the sex difference in chicken muscle development.

Neutrophil and natural killer cell imbalances prevent muscle stem cell mediated regeneration following murine volumetric muscle loss

Volumetric muscle loss (VML) overwhelms the innate regenerative capacity of mammalian skeletal muscle (SkM), leading to numerous disabilities and reduced quality of life. Immune cells are critical responders to muscle injury and guide tissue resident stem cell and progenitor mediated myogenic repair. However, how immune cell infiltration and inter-cellular communication networks with muscle stem cells are altered following VML and drive pathological outcomes remains underexplored. Herein, we contrast the cellular and molecular mechanisms of VML injuries that result in fibrotic degeneration or regeneration of SkM. Following degenerative VML injuries, we observe heightened infiltration of natural killer (NK) cells as well as persistence of neutrophils beyond two weeks post injury. Functional validation of NK cells revealed an antagonistic role on neutrophil accumulation in part via inducing apoptosis and CCR1 mediated chemotaxis. The persistent infiltration of neutrophils in degenerative VML injuries was found to contribute to impairments in muscle stem cell regenerative function, which was also attenuated by transforming growth factor beta 1 (TGFb1). Blocking TGFb signaling reduced neutrophil accumulation and fibrosis, as well as improved muscle specific force. Collectively, these results enhance our understanding of immune cell-stem cell crosstalk that drives regenerative dysfunction and provide further insight into possible avenues for fibrotic therapy exploration.

Myosin-based regulation of twitch and tetanic contractions in mammalian skeletal muscle

Time-resolved X-ray diffraction from isolated fast-twitch muscles of the mouse was used to show how structural changes in the myosin-containing thick filaments contribute to the regulation of muscle contraction, extending the previous focus on regulation by the actin-containing thin filaments. This study shows that muscle activation involves the following sequence of structural changes: thin filament activation, disruption of the helical array of myosin motors characteristic of resting muscle, release of myosin motor domains from the folded conformation on the filament backbone, and actin attachment. Physiological force generation in the 'twitch' response of skeletal muscle to single action potential stimulation is limited by incomplete activation of the thick filament and the rapid inactivation of both filaments. Muscle relaxation after repetitive stimulation is accompanied by complete recovery of the folded motor conformation on the filament backbone but incomplete reformation of the helical array, revealing a structural basis for post-tetanic potentiation in isolated muscle.

Skeletal Muscles: Insight into Embryonic Development, Satellite Cells, Histology, Ultrastructure, Innervation, Contraction and Relaxation, Causes, Pathophysiology, and Treatment of Volumetric Muscle I

Background: Skeletal muscles are attached to bone and are responsible for the axial and appendicular movement of the skeleton and for maintenance of body position and posture. Objectives: The present review aimed to high light on embryonic development of skeletal muscles, histological and ultrastructure, innervation, contraction and relaxation, causes, pathophysiology, and treatment of volumetric muscle injury. The heterogeneity of the muscle fibers is the base of the flexibility which allows the same muscle to be used for various tasks from continuous low-intensity activity, to repeated submaximal contractions, and to fast and strong maximal contractions. The formation of skeletal muscle begins during the fourth week of embryonic development as specialized mesodermal cells, termed myoblasts. As growth of the muscle fibers continues, aggregation into bundles occurs, and by birth, myoblast activity has ceased. Satellite cells (SCs), have single nuclei and act as regenerative cells. Satellite cells are the resident stem cells of skeletal muscle; they are considered to be self-renewing and serve to generate a population of differentiation-competent myoblasts that will participate as needed in muscle growth, repair, and regeneration. Based on various structural and functional characteristics, skeletal muscle fibres are classified into three types: Type I fibres, Type II-B fibres, and type II-A fibres. Skeletal muscle fibres vary in colour depending on their content of myoglobin. Each myofibril exhibits a repeating pattern of cross-striations which is a product of the highly ordered arrangement of the contractile proteins within it. The parallel myofibrils are arranged with their cross-striations in the register, giving rise to the regular striations seen with light microscopy in longitudinal sections of skeletal muscle. Each skeletal muscle receives at least two types of nerve fibers: motor and sensory. Striated muscles and myotendinous junctions contain sensory receptors that are encapsulated proprioceptors. The process of contraction, usually triggered by neural impulses, obeys the all-or-none law. During muscle contraction, the thin filaments slide past the thick filaments, as proposed by Huxley's sliding filament theory. In response to a muscle injury, SCs are activated and start to proliferate; at this stage, they are often referred to as either myogenic precursor cells (MPC) or myoblasts. In vitro, evidence has been presented that satellite cells can be pushed towards the adipogenic and osteogenic lineages, but contamination of such cultures from non-myogenic cells is sometimes hard to dismiss as the underlying cause of this observed multipotency. There are, however, other populations of progenitors isolated from skeletal muscle, including endothelial cells and muscle-derived stem cells (MDSCs), blood-vessel-associated mesoangioblasts, muscle side-population cells, CD133+ve cells, myoendothelial cells, and pericytes. Volumetric muscle loss (VML) is defined as the traumatic or surgical loss of skeletal muscle with resultant functional impairment. It represents a challenging clinical problem for both military and civilian medicine. VML results in severe cosmetic deformities and debilitating functional loss. In response to damage, skeletal muscle goes through a well-defined series of events including; degeneration (1 to 3days), inflammation, and regeneration (3 to 4 weeks), fibrosis, and extracellular matrix remodeling (3 to 6 months).. Mammalian skeletal muscle has an impressive ability to regenerate itself in response to injury. During muscle tissue repair following damage, the degree of damage and the interactions between muscle and the infiltrating inflammatory cells appear to affect the successful outcome of the muscle repair process. The transplantation of stem cells into aberrant or injured tissue has long been a central goal of regenerative medicine and tissue engineering. Conclusion: It can be concluded that the formation of skeletal muscle begins during the fourth week of embryonic development as specialized mesodermal cells, termed myoblasts, by birth myoblast activity has ceased. Satellite cells are considered to be self-renewing, and serve to generate a population of differentiation-competent myoblasts. Skeletal muscle fibres are classified into three types. The process of contraction, usually triggered by neural impulses, obeys the all-or-none law. VML results in severe cosmetic deformities and debilitating functional loss. Mammalian skeletal muscle has an impressive ability to regenerate itself in response to injury. The transplantation of stem cells into aberrant or injured tissue has long been a central goal of regenerative medicine and tissue engineering.

Time-resolved transcriptional profiling of Trichinella-infected murine myocytes helps to elucidate host–pathogen interactions in the muscle stage

Background Parasites of the genus Trichinella are the pathogenic agents of trichinellosis, which is a widespread and severe foodborne parasitic disease. Trichinella spiralis resides primarily in mammalian skeletal muscle cells. After invading the cells of the host organism, T. spiralis must elude or invalidate the host’s innate and adaptive immune responses to survive. It is necessary to characterize the pathogenesis of trichinellosis to help to prevent the occurrence and further progression of this disease. The aims of this study were to elucidate the mechanisms of nurse cell formation, pathogenesis and immune evasion of T. spiralis, to provide valuable information for further research investigating the basic cell biology of Trichinella-infected muscle cells and the interaction between T. spiralis and its host. Methods We performed transcriptome profiling by RNA sequencing to identify global changes at 1, 3, 7, 10 and 15 days post-infection (dpi) in gene expression in the diaphragm after the parasite entered and persisted within the murine myocytes; the mice were infected by intravenous injection of newborn larvae. Gene expression analysis was based on the alignment results. Differentially expressed genes (DEGs) were identified based on their expression levels in various samples, and functional annotation and enrichment analysis were performed. Results The most extensive and dynamic gene expression responses in host diaphragms were observed during early infection (1 dpi). The number of DEGs and genes annotated in the Kyoto Encyclopedia of Genes and Genomes and Gene Ontology databases decreased significantly in the infected mice compared to the uninfected mice at 3 and 7 dpi, suddenly increased sharply at 10 dpi, and then decreased to a lower level at 15 dpi, similar to that observed at 3 and 7 dpi. The massive initial reaction of the murine muscle cells to Trichinella infection steadied in the later stages of infection, with little additional changes detected for the remaining duration of the studied process. Although there were hundreds of DEGs at each time point, only 11 genes were consistently up- or downregulated at all 5 time points. Conclusions The gene expression patterns identified in this study can be employed to characterize the coordinated response of T. spiralis-infected myocytes in a time-resolved manner. This comprehensive dataset presents a distinct and sensitive picture of the interaction between host and parasite during intracellular infection, which can help to elucidate how pathogens evade host defenses and coordinate the biological functions of host cells to survive in the mammalian environment.