Metabolome in Tibialis and Soleus Muscles in Wild-Type and Pin1 Knockout Mice through High-Resolution Magic Angle Spinning 1H Nuclear Magnetic Resonance Spectroscopy.

Skeletal muscles are heterogenous tissues composed of different myofiber types that can be classified as slow oxidative, fast oxidative, and fast glycolytic which are distinguished on the basis of their contractile and metabolic properties. Improving oxidative metabolism in skeletal muscles can prevent metabolic diseases and plays a protective role against muscle wasting in a number of neuromuscular diseases. Therefore, achieving a detailed understanding of the factors that regulate myofiber metabolic properties might provide new therapeutic opportunities for these diseases. Here, we investigated whether peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (PIN1) is involved in the control of myofiber metabolic behaviors. Indeed, PIN1 controls glucose and lipid metabolism in a number of tissues, and it is also abundant in adult skeletal muscles; however, its role in the control of energy homeostasis in this tissue is still to be defined. To start clarifying this topic, we compared the metabolome of the tibialis anterior muscle (mainly glycolytic) and soleus muscle (oxidative) in wild-type and Pin1 knockout mice with High-Resolution Magic Angle Spinning (HR-MAS) NMR on intact tissues. Our analysis reveals a clear demarcation between the metabolomes in the two types of muscles and allows us to decode a signature able to discriminate the glycolytic versus oxidative muscle phenotype. We also detected some changes in Pin1-depleted muscles that suggest a role for PIN1 in regulating the metabolic phenotype of skeletal muscles.

Inflammatory-Targeted Lipid Carrier as a New Nanomaterial to Formulate an Inhaled Drug Delivery System.

There is a pressing need for efficacious therapies in the field of respiratory diseases and infections. Lipid nanocarriers, administered through aerosols, represent a promising tool for maximizing therapeutic concentration in targeted cells and minimizing systemic exposure. However, this approach requires the application of efficient and safe nanomaterials. Palmitoylethanolamide (PEA), an endocannabinoid-like endogenous lipid, plays a crucial role in providing protective mechanisms during inflammation, making it an interesting material for preparing inhalable lipid nanoparticles (LNPs). This report aims to preliminarily explore the in vitro behavior of LNPs prepared with PEA (PEA-LNPs), a new inhalable inflammatory-targeted nanoparticulate drug carrier. PEA-LNPs exhibited a size of about 250 nm, a rounded shape, and an marked improvement in PEA solubility in comparison to naked PEA, indicative of easily disassembled nanoparticles. A twin glass impinger instrument was used to screen the aerosol performance of PEA-LNP powders, obtained via freeze-drying in the presence of two quantities of mannose as a cryoprotectant. Results indicated that a higher amount of mannose improved the emitted dose (ED), and in particular, the fine particle fraction (FPF). A cytotoxicity assay was performed and indicated that PEA-LNPs are not toxic towards the MH-S alveolar macrophage cell line up to concentrations of 0.64 mg/mL, and using coumarin-6 labelled particles, a rapid internalization into the macrophage was confirmed. This study demonstrates that PEA could represent a suitable material for preparing inhalable lipid nanocarrier-based dry powders, which signify a promising tool for the transport of drugs employed to treat respiratory diseases and infections.

Epitranscriptomics as a New Layer of Regulation of Gene Expression in Skeletal Muscle: Known Functions and Future Perspectives.

Epitranscriptomics refers to post-transcriptional regulation of gene expression via RNA modifications and editing that affect RNA functions. Many kinds of modifications of mRNA have been described, among which are N6-methyladenosine (m6A), N1-methyladenosine (m1A), 7-methylguanosine (m7G), pseudouridine (Ψ), and 5-methylcytidine (m5C). They alter mRNA structure and consequently stability, localization and translation efficiency. Perturbation of the epitranscriptome is associated with human diseases, thus opening the opportunity for potential manipulations as a therapeutic approach. In this review, we aim to provide an overview of the functional roles of epitranscriptomic marks in the skeletal muscle system, in particular in embryonic myogenesis, muscle cell differentiation and muscle homeostasis processes. Further, we explored high-throughput epitranscriptome sequencing data to identify RNA chemical modifications in muscle-specific genes and we discuss the possible functional role and the potential therapeutic applications.

Corrigendum: Histone deacetylase functions and therapeutic implications for adult skeletal muscle metabolism.

[This corrects the article DOI: 10.3389/fmolb.2023.1130183.].

Histone deacetylase functions and therapeutic implications for adult skeletal muscle metabolism.

Skeletal muscle is a highly adaptive organ that sustains continuous metabolic changes in response to different functional demands. Healthy skeletal muscle can adjust fuel utilization to the intensity of muscle activity, the availability of nutrients and the intrinsic characteristics of muscle fibers. This property is defined as metabolic flexibility. Importantly, impaired metabolic flexibility has been associated with, and likely contributes to the onset and progression of numerous pathologies, including sarcopenia and type 2 diabetes. Numerous studies involving genetic and pharmacological manipulations of histone deacetylases (HDACs) in vitro and in vivo have elucidated their multiple functions in regulating adult skeletal muscle metabolism and adaptation. Here, we briefly review HDAC classification and skeletal muscle metabolism in physiological conditions and upon metabolic stimuli. We then discuss HDAC functions in regulating skeletal muscle metabolism at baseline and following exercise. Finally, we give an overview of the literature regarding the activity of HDACs in skeletal muscle aging and their potential as therapeutic targets for the treatment of insulin resistance.

Design, Characterization, and In Vitro Assays on Muscle Cells of Endocannabinoid-like Molecule Loaded Lipid Nanoparticles for a Therapeutic Anti-Inflammatory Approach to Sarcopenia.

Inflammatory processes play a key role in the pathogenesis of sarcopenia owing to their effects on the balance between muscle protein breakdown and synthesis. Palmitoylethanolamide (PEA), an endocannabinoid-like molecule, has been well documented for its anti-inflammatory properties, suggesting its possible beneficial use to counteract sarcopenia. The promising therapeutic effects of PEA are, however, impaired by its poor bioavailability. In order to overcome this limitation, the present study focused on the encapsulation of PEA in solid lipid nanoparticles (PEA-SLNs) in a perspective of a systemic administration. PEA-SLNs were characterized for their physico-chemical properties as well as cytotoxicity and cell internalization capacity on C2C12 myoblast cells. Their size was approximately 250 nm and the encapsulation efficiency reached 90%. Differential scanning calorimetry analyses demonstrated the amorphous state of PEA in the inner SLN matrix, which improved PEA dissolution, as observed in the in vitro assays. Despite the high internalization capacity observed with the flow cytometer (values between 85 and 94% after 14 h of incubation), the Nile Red labeled PEA-SLNs showed practically no toxicity towards myoblasts. Confocal analysis showed the presence of SLNs in the cytoplasm and not in the nucleus. These results suggest the potentiality provided by PEA-SLNs to obtain an innovative and side-effect-free tool in the medical treatment of sarcopenia.

The transcription factor NF-Y participates to stem cell fate decision and regeneration in adult skeletal muscle.

The transcription factor NF-Y promotes cell proliferation and its activity often declines during differentiation through the regulation of NF-YA, the DNA binding subunit of the complex. In stem cell compartments, the shorter NF-YA splice variant is abundantly expressed and sustains their expansion. Here, we report that satellite cells, the stem cell population of adult skeletal muscle necessary for its growth and regeneration, express uniquely the longer NF-YA isoform, majorly associated with cell differentiation. Through the generation of a conditional knock out mouse model that selectively deletes the NF-YA gene in satellite cells, we demonstrate that NF-YA expression is fundamental to preserve the pool of muscle stem cells and ensures robust regenerative response to muscle injury. In vivo and ex vivo, satellite cells that survive to NF-YA loss exit the quiescence and are rapidly committed to early differentiation, despite delayed in the progression towards later states. In vitro results demonstrate that NF-YA-depleted muscle stem cells accumulate DNA damage and cannot properly differentiate. These data highlight a new scenario in stem cell biology for NF-Y activity, which is required for efficient myogenic differentiation.

Alternative Splicing of Transcription Factors Genes in Muscle Physiology and Pathology.

Skeletal muscle formation is a multi-step process that is governed by complex networks of transcription factors. The regulation of their functions is in turn multifaceted, including several mechanisms, among them alternative splicing (AS) plays a primary role. On the other hand, altered AS has a role in the pathogenesis of numerous muscular pathologies. Despite these premises, the causal role played by the altered splicing pattern of transcripts encoding myogenic transcription factors in neuromuscular diseases has been neglected so far. In this review, we systematically investigate what has been described about the AS patterns of transcription factors both in the physiology of the skeletal muscle formation process and in neuromuscular diseases, in the hope that this may be useful in re-evaluating the potential role of altered splicing of transcription factors in such diseases.

Dynamic Phosphorylation of the Myocyte Enhancer Factor 2Cα1 Splice Variant Promotes Skeletal Muscle Regeneration and Hypertrophy.

The transcription factor MEF2C (Myocyte Enhancer Factor 2C) plays an established role in the early steps of myogenic differentiation. However, the involvement of MEF2C in adult myogenesis and in muscle regeneration has not yet been systematically investigated. Alternative splicing of mammalian MEF2C transcripts gives rise to two mutually exclusive protein variants: MEF2Cα2 which exerts a positive control of myogenic differentiation, and MEF2Cα1, in which the α1 domain acts as trans-repressor of the MEF2C pro-differentiation activity itself. However, MEF2Cα1 variants are persistently expressed in differentiating cultured myocytes, suggesting a role in adult myogenesis. We found that overexpression of both MEF2Cα1/α2 proteins in a mouse model of muscle injury promotes muscle regeneration and hypertrophy, with each isoform promoting different stages of myogenesis. Besides the ability of MEF2Cα2 to increase differentiation, we found that overexpressed MEF2Cα1 enhances both proliferation and differentiation of primary myoblasts, and activates the AKT/mTOR/S6K anabolic signaling pathway in newly formed myofibers. The multiple activities of MEF2Cα1 are modulated by phosphorylation of Ser98 and Ser110, two amino acid residues located in the α1 domain of MEF2Cα1. These specific phosphorylations allow the interaction of MEF2Cα1 with the peptidyl-prolyl isomerase PIN1, a regulator of MEF2C functions. Overall, in this study we established a novel regulatory mechanism in which the expression and the phosphorylation of MEF2Cα1 are critically required to sustain the adult myogenesis. The described molecular mechanism will represent a new potential target for the development of therapeutical strategies to treat muscle-wasting diseases. Stem Cells 2017;35:725-738.

NF-YA splice variants have different roles on muscle differentiation.

The heterotrimeric CCAAT-binding factor NF-Y controls the expression of a multitude of genes involved in cell cycle progression. NF-YA is present in two alternatively spliced isoforms, NF-YAs and NF-YAl, differing in 28 aminoacids in the N-terminal Q-rich activation domain. NF-YAs has been identified as a regulator of stemness and proliferation in mouse embryonic cells (mESCs) and human hematopoietic stem cells (hHSCs), whereas the role of NF-YAl is not clear. In the muscle system, NF-YA expression is observed in proliferating cells, but barely detectable in terminally differentiated cells in vitro and adult skeletal muscle in vivo. Here, we show that NF-YA inactivation in mouse myoblasts impairs both proliferation and differentiation. The overexpression of the two NF-YA isoforms differentially affects myoblasts fate: NF-YAs enhance cell proliferation, while NF-YAl boosts differentiation. The molecular mechanisms were investigated by expression profilings, detailing the opposite programs of the two isoforms. Bioinformatic analysis of the regulated promoters failed to detect a significant presence of CCAAT boxes in the regulated genes. NF-YAl activates directly Mef2D, Six genes, and p57kip2 (Cdkn1c), and indirectly the myogenic regulatory factors (MRFs). Specifically, Cdkn1c activation is induced by NF-Y binding to its CCAAT promoter and by reducing the expression of the lncRNA Kcnq1ot1, a negative regulator of Cdkn1c transcription. Overall, our results indicate that NF-YA alternative splicing is an influential muscle cell determinant, through direct regulation of selected cell cycle blocking genes, and, directly and indirectly, of muscle-specific transcription factors.

Phosphorylation-dependent degradation of MEF2C contributes to regulate G2/M transition.

The Myocyte Enhancer Factor 2C (MEF2C) transcription factor plays a critical role in skeletal muscle differentiation, promoting muscle-specific gene transcription. Here we report that in proliferating cells MEF2C is degraded in mitosis by the Anaphase Promoting Complex/Cyclosome (APC/C) and that this downregulation is necessary for an efficient progression of the cell cycle. We show that this mechanism of degradation requires the presence on MEF2C of a D-box (R-X-X-L) and 2 phospho-motifs, pSer98 and pSer110. Both the D-box and pSer110 motifs are encoded by the ubiquitous alternate α1 exon. These two domains mediate the interaction between MEF2C and CDC20, a co-activator of APC/C. We further report that in myoblasts, MEF2C regulates the expression of G2/M checkpoint genes (14-3-3γ, Gadd45b and p21) and the sub-cellular localization of CYCLIN B1. The importance of controlling MEF2C levels during the cell cycle is reinforced by the observation that modulation of its expression affects the proliferation rate of colon cancer cells. Our findings show that beside the well-established role as pro-myogenic transcription factor, MEF2C can also function as a regulator of cell proliferation.

Proline isomerase Pin1 represses terminal differentiation and myocyte enhancer factor 2C function in skeletal muscle cells.

Reversible proline-directed phosphorylation at Ser/Thr-Pro motifs has an essential role in myogenesis, a multistep process strictly regulated by several signaling pathways that impinge on two families of myogenic effectors, the basic helix-loop-helix myogenic transcription factors and the MEF2 (myocyte enhancer factor 2) proteins. The question of how these signals are deciphered by the myogenic effectors remains largely unaddressed. In this study, we show that the peptidyl-prolyl isomerase Pin1, which catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds to induce conformational changes of its target proteins, acts as an inhibitor of muscle differentiation because its knockdown in myoblasts promotes myotube formation. With the aim of clarifying the mechanism of Pin1 function in skeletal myogenesis, we investigated whether MEF2C, a critical regulator of the myogenic program that is the end point of several signaling pathways, might serve as a/the target for the inhibitory effects of Pin1 on muscle differentiation. We show that Pin1 interacts selectively with phosphorylated MEF2C in skeletal muscle cells, both in vitro and in vivo. The interaction with Pin1 requires two novel critical phospho-Ser/Thr-Pro motifs in MEF2C, Ser(98) and Ser(110), which are phosphorylated in vivo. Overexpression of Pin1 decreases MEF2C stability and activity and its ability to cooperate with MyoD to activate myogenic conversion. Collectively, these findings reveal a novel role for Pin1 as a regulator of muscle terminal differentiation and suggest that Pin1-mediated repression of MEF2C function could contribute to this function.

Differentiation-dependent lysine 4 acetylation enhances MEF2C binding to DNA in skeletal muscle cells.

Myocyte enhancer factor 2 (MEF2) proteins play a key role in promoting the expression of muscle-specific genes in differentiated muscle cells. MEF2 activity is regulated by the association with several transcriptional co-factors and by post-translational modifications. In the present report, we provide evidence for a novel regulatory mechanism of MEF2C activity, which occurs at the onset of skeletal muscle differentiation and is based on Lys4 acetylation. This covalent modification results in the enhancement of MEF2C binding to DNA and chromatin. In particular, we report that the kinetic parameters of MEF2/DNA association change substantially upon induction of differentiation to give a more stable complex and that this effect is mediated by Lys4 acetylation. We also show that Lys4 acetylation plays a prominent role in the p300-dependent activation of MEF2C.

Low-density lipoprotein (LDL) receptor/transferrin fusion protein: in vivo production and functional evaluation as a potential therapeutic tool for lowering plasma LDL cholesterol.

A soluble form of human low-density lipoprotein receptor (LDL-R) fused in frame with rabbit transferrin (LDL-Rs(hu)/Tf(rab)) is assessed in vivo as a therapeutic tool for lowering plasma LDL cholesterol. The cDNA encoding LDL-Rs(hu)/Tf(rab) is expressed in mice, using a hydrodynamics-based gene transfer procedure. The transgene is transcribed in the liver of transduced animals and the corresponding protein is secreted into the bloodstream. Circulating LDL-Rs(hu)/Tf(rab) binds LDL specifically, thus indicating that it is correctly processed through the cellular compartments in vivo. More importantly, the expression of LDL-Rs(hu)/Tf(rab) allows the removal of injected human (125)I-labeled LDL ((123)I-LDL) from the bloodstream of transduced CD1 mice, which show faster LDL plasma clearance, anticipating by approximately 90 min the same clearance value observed in control animals. A similar effect is observed in transduced LDL-R(-/-) mice, in which the clearance of injected human LDL depends solely on the presence of circulating LDL-Rs(hu) /Tf(rab). In these animals the extent of plasma LDL clearance is directly related to the concentration of LDL-Rs(hu)/Tf(rab) in the blood. Finally, LDL-Rs(hu)/Tf(rab) does not alter the pattern of LDL organ distribution: in transduced animals, as well as in control animals, liver and bladder are the predominantly labeled organs after (123)I-LDL injection. However, LDL-Rs(hu)/Tf(rab) has a quantitative effect on LDL tissue deposition: in treated animals LDL-Rs(hu)/Tf(rab) determines an increase in radioactivity in the liver at early times after (123)I-LDL injection and a progressive labeling of the bladder, starting 20 min after injection.