Skeletal muscle capillarization is proportional to muscle fibre mitochondrial content and oxidative capacity. Skeletal muscle cells secrete many factors that regulate neighbouring capillary endothelial cells (ECs), including extracellular vesicles (SkM-EVs). Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1 alpha) regulates mitochondrial biogenesis and the oxidative phenotype in skeletal muscle. Skeletal muscle PGC-1 alpha also regulates secretion of multiple angiogenic factors, but it is unknown whether PGC-1 alpha regulates SkM-EV release, contents and angiogenic signalling potential. PGC-1 alpha was overexpressed via adenovirus in primary human myotubes. EVs were collected from PGC-1 alpha-overexpressing myotubes (PGC-EVs) as well as from green fluorescent protein-overexpressing myotubes (GFP-EVs), and from untreated myotubes. EV release and select mRNA contents were measured from EVs. Additionally, ECs were treated with EVs to measure angiogenic potential of EVs in normal conditions and following an oxidative stress challenge. PGC-1 alpha overexpression did not impact EV release but did elevate EV content of mRNAs for several antioxidant proteins (nuclear factor erythroid 2-related factor 2, superoxide dismutase 2, glutathione peroxidase). PGC-EV treatment of cultured human umbilical vein endothelial cells (HUVECs) increased their proliferation (+36.6%), tube formation (length: +28.1%; number: +25.7%) and cellular viability (+52.9%), and reduced reactive oxygen species levels (-41%) compared to GFP-EVs. Additionally, PGC-EV treatment protected against tube formation impairments and induction of cellular senescence following acute oxidative stress. Overexpression of PGC-1 alpha in human myotubes increases the angiogenic potential of SkM-EVs. These angiogenic benefits coincided with increased anti-oxidative capacity of recipient HUVECs. High PGC-1 alpha expression in skeletal muscle may prompt the release of SkM-EVs that support vascular redox homeostasis and angiogenesis.

The lipid droplet (LD) is a central hub for fatty acid metabolism in cells. Here we define the dynamics and explore the role of LDs in skeletal muscle satellite cells (SCs), a stem cell population responsible for muscle regeneration. In newly divided SCs, LDs are unequally distributed in sister cells exhibiting asymmetric cell fates, as the LDLow cell self-renews while the LDHigh cell commits to differentiation. When transplanted into regenerating muscles, LDLow cells outperform LDHigh cells in self-renewal and regeneration in vivo. Pharmacological inhibition of LD biogenesis or genetic inhibition of LD catabolism through knockout of Pnpla2 (encoding ATGL, the rate-limiting enzyme for lipolysis) disrupts cell fate homeostasis and impairs the regenerative capacity of SCs. Dysfunction of Pnpla2-null SCs is associated with energy insufficiency and oxidative stress that can be partially rescued by antioxidant (N-acetylcysteine) treatment. These results establish a direct link between LD dynamics and stem cell fate determination.

Mitochondrial remodeling through fusion and fission is crucial for progenitor cell differentiation but its role in myogenesis is poorly understood. Here, we characterized the function of mitofusin 2 (Mfn2), a mitochondrial outer membrane protein critical for mitochondrial fusion, in muscle progenitor cells (myoblasts). Mfn2 expression is upregulated during myoblast differentiation in vitro and muscle regeneration in vivo. Targeted deletion of Mfn2 gene in myoblasts (Mfn2(MKO)) increases oxygen-consumption rates (OCR) associated with the maximal respiration and spare respiratory capacity, and increased levels of reactive oxygen species (ROS). Skeletal muscles of Mfn2(MKO) mice exhibit robust mitochondrial swelling with normal mitochondrial DNA content. Additionally, mitochondria isolated from Mfn2(MKO) muscles have reduced OCR at basal state and for complex I respiration, associated with decreased levels of complex I proteins NDUFB8 (NADH ubiquinone oxidoreductase subunit B8) and NDUFS3 (NADH ubiquinone oxidoreductase subunit S3). However, Mfn2(MKO) has no obvious effects on myoblast differentiation, muscle development and function, and muscle regeneration. These results demonstrate a novel role of Mfn2 in regulating mitochondrial complex I protein abundance and respiratory functions in myogenic progenitors and myofibers.

Unraveling the complexity of the lipidome requires the development of novel approaches for the structural characterization of lipid species with isomer-level discrimination. Herein, we introduce an online photochemical approach for lipid isomer identification through selective derivatization of double bonds by reaction with singlet oxygen. Lipid hydroperoxide products are generated promptly after laser irradiation. Fragmentation of these species in a mass spectrometer produces diagnostic fragments revealing the C=C locations in the unreacted lipids. This approach uses an inexpensive light source and photosensitizer making it easy to incorporate into any lipidomics workflow. We demonstrate the utility of this approach for the shotgun profiling of C=C locations in different lipid classes present in tissue extracts using electrospray ionization (ESI) and ambient imaging of lipid species differing only by the location of C=C bonds using nanospray desorption electrospray ionization (nano-DESI).

Duchenne muscular dystrophy (DMD) is caused by a mutation of the muscle membrane protein dystrophin and characterized by severe degeneration of myofibers, progressive muscle wasting, loss of mobility, and, ultimately, cardiorespiratory failure and premature death. Currently there is no cure for DMD. Herein, we report that skeletal muscle-specific knockout (KO) of the phosphatase and tensin homolog (Pten) gene in an animal model of DMD (mdx mice) alleviates myofiber degeneration and restores muscle function without increasing tumor incidence. Specifically, Pten KO normalizes myofiber size and prevents muscular atrophy, and it improves grip strength and exercise performance in mdx mice. Pten KO also reduces fibrosis and inflammation, and it ameliorates muscle pathology in mdx mice. Unbiased RNA sequencing reveals that Pten KO upregulates extracellular matrix and basement membrane components positively correlated with wound healing and suppresses negative regulators of wound healing and lipid biosynthesis, thus improving the integrity of muscle basement membrane at the ultrastructural level. Importantly, pharmacological inhibition of PTEN similarly ameliorates muscle pathology and improves muscle integrity and function in mdx mice. Our findings provide evidence that PTEN inhibition may represent a potential therapeutic strategy to restore muscle function in DMD.

Myogenesis includes sequential stages of progenitor cell proliferation, myogenic commitment and differentiation, myocyte fusion, and myotube maturation. Different stages of myogenesis are orchestrated and regulated by myogenic regulatory factors and various downstream cellular signaling. Here we identify phosphatase orphan 1 (Phospho1) as a new player in myogenesis. During activation, proliferation, and differentiation of quiescent satellite cells, the expression of Phospho1 gradually increases. Overexpression of Phospho1 inhibits myoblast proliferation but promotes their differentiation and fusion. Conversely, knockdown of Phospho1 accelerates myoblast proliferation but impairs myotube formation. Moreover, knockdown of Phospho1 decreases the OXPHO protein levels and mitochondria density, whereas overexpression of Phospho1 upregulates OXPHO protein levels and promotes mitochondrial oxygen consumption. Finally, we show that Phospho1 expression is controlled by myogenin, which binds to the promoter of Phospho1 to regulate its transcription. These results indicate a key role of Phospho1 in regulating myogenic differentiation and mitochondrial function.

Mammalian skeletal muscle possesses a unique ability to regenerate, which is primarily mediated by a population of resident muscle stem cells (MuSCs) and requires a concerted response from other supporting cell populations. Previous targeted analysis has described the involvement of various specific populations in regeneration, but an unbiased and simultaneous evaluation of all cell populations has been limited. Therefore, we used single-cell RNA-sequencing to uncover gene expression signatures of over 53,000 individual cells during skeletal muscle regeneration. Cells clustered into 25 populations and subpopulations, including a subpopulation of immune gene enriched myoblasts (immunomyoblasts) and subpopulations of fibro-adipogenic progenitors. Our analyses also uncovered striking spatiotemporal dynamics in gene expression, population composition, and cell-cell interaction during muscle regeneration. These findings provide insights into the cellular and molecular underpinning of skeletal muscle regeneration.

Nonpolar triglycerides (TGs) are rarely detected in mass spectrometry imaging (MSI) experiments unless they are abundant in the sample. Herein, we use nanospray desorption electrospray ionization (nano-DESI) to explore the role of the solvent composition and ionic dopants on the detection of TGs in a murine gastrocnemius muscle tissue used as a model system. We evaluated three solvent mixtures for their ability to extract nonpolar TG species: MeOH:H2O 9:1 (v/v), MeOH:DCM 6:4 (v/v) and MeO-H:AcN:tol 5:3.5:1.5 (v/v/v). We observe that TGs are mainly detected as [M+K](+) adducts and their extraction efficiency is improved using less polar solvents: MeOH:DCM and MeOH:AcN:tol. We also explore whether the ionization efficiency of TGs may be improved by doping the MeOH:AcN:tol solvent with ammonium formate (AF) and other ionic additives. However, the formation of [M+NH4](+) adducts of TGs is less efficient than that of [M+K](+) adducts in the range of AF concentrations from 0.1 to 10 mM. Chemical derivatization using 100 mu M of Girard T reagent predominately generates reaction products of phosphatidylcholines rather than TG species. Moreover, the presence of the Girard T reagent suppresses ion signals of all the species in the spectrum including TGs. Nano-DESI MSI experiments performed using MeOH:AcN:tol solvent enable imaging of TGs without any detectable adverse effect on signals of other lipids and metabolites. Specifically, 10 out of 14 TG species were detected exclusively using MeOH:AcN:tol and the sensitivity towards other TGs was improved by at least an order of magnitude. Although polyunsaturated TGs can be detected using both solvents, saturated and monounsaturated TGs are only detected using MeOH:AcN:tol. Our results provide a direct path for the improved detection of TGs in tissue imaging experiments using liquid-based ambient ionization techniques. (C) 2019 Elsevier B.V. All rights reserved.

Skeletal muscles contain heterogeneous myofibers that are different in size and contractile speed, with type IIb myofiber being the largest and fastest. Here, we identify methyltransferase-like 21e (Mettl21e), a member of newly classified nonhistone methyltransferases, as a gene enriched in type IIb myofibers. The expression of Mettl21e was strikingly up-regulated in hypertrophic muscles and during myogenic differentiation in vitro and in vivo. Knockdown (KD) of Mettl21e led to atrophy of cultured myotubes, and targeted mutation of Mettl21e in mice reduced the size of IIb myofibers without affecting the composition of myofiber types. Mass spectrometry and methyltransferase assay revealed that Mettl21e methylated valosin-containing protein (Vcp/p97), a key component of the ubiquitin-proteasome system. KD or knockout of Mettl21e resulted in elevated 26S proteasome activity, and inhibition of proteasome activity prevented atrophy of Mettl21e KD myotubes. These results demonstrate that Mettl21e functions to maintain myofiber size through inhibiting proteasome-mediated protein degradation.-Wang, C., Zhang, B., Ratliff, A. C., Arrington, J., Chen, J., Xiong, Y., Yue, F., Nie, Y., Hu, K., Jin, W., Tao, W. A., Hrycyna, C. A., Sun, X., Kuang, S. Methyltransferase-like 21e inhibits 26S proteasome activity to facilitate hypertrophy of type IIb myofibers.