Notch信号是一种进化保守且能调节多种细胞发育的信号通路,在发育过程中其对细胞间的通讯和细胞命运的决定具有重要意义,也是组织稳态所必需的。Notch的配体(Dll1、3、4和Jag1、2)和受体(Notch1-4)均为跨膜蛋白,经典的Notch激活方式是当膜上的配体与相邻受体结合后会促使受体胞内结构域剪切入核,作为转录共激活子与RBPJ共同激活下游靶基因的转录。大量的研究已表明,Notch信号通路在早期肌节和四肢骨骼肌的发育过程中均具有重要的作用;在胎儿期,Notch可以调控肌肉干细胞定位到基质膜与肌纤维膜之间形成出生后再生过程所必须的肌卫星细胞(SCs);而在成年期,Notch调控着肌卫星细胞静息状态的维持、自我更新和分化的平衡。虽然在肌肉发育过程中Notch信号的作用已经明确,但在活体水平具体是来自何种肌肉细胞的哪一种配体主要调控Notch信号及肌肉干细胞功能还不明确。因此本试验首先通过分析肌肉组织和肌肉干细胞数据库解析了5个配体在肌肉中的表达模式以及表达水平,并通过实验验证其表达,确定了Dll1是肌肉发育过程中高表达的配体。接着,通过制备Myo DCre、MLCCre以及Pax7Cre ER驱动的Dll1肌肉细胞特异性敲除鼠,探索来自哪类肌细胞的Dll1对肌肉的发育和再生产生影响。随后,通过成肌细胞及肌纤维培养、活体追踪等实验进一步明确了Dll1对肌肉干细胞命运的调控。最后,探索了在肌肉干细胞中Dll1调控Notch信号的作用方式。获得主要结果如下:1.对已有的肌肉相关测序数据分析显示,在肌肉组织和肌肉干细胞中配体Dll1和Jag1的表达水平较高;在成肌分化过程中Dll1、Jag2和Dll4表达水平显著上升,Jag1变化波动不明显。2.通过cre-loxp系统构建了MLCCre-Dll1(Dll1MLCKO)敲除鼠。分析肌肉显示,肌纤维中敲除Dll1并不影响肌肉的发育;肌纤维中敲除Dll1并不能显著影响肌肉的再生且对SCs的数目无显著调节;跑步测试实验证明肌纤维中敲除Dll1不影响肌肉的运动能力。3.Myo DCre-Dll1(Dll1Myo DKO)敲除鼠在15天左右出先胚胎死亡。肌肉细胞中敲除Dll1导致了严重肌肉发育不良,并且出现肌肉干细胞的严重缺失。进一步研究发现Dll1Myo DKO敲除鼠从E11.5天开始出现肌节和四肢骨骼肌肌肉干细胞的消耗。这一过程伴随着Myo G阳性细胞数比例的瞬时上调。4.Pax7Cre ER-Dll1(Dll1Pax7KO)敲除鼠在出生后诱导Dll1敲除,下调了肌肉重量及肌纤维大小,同时伴随着肌肉干细胞数目的显著下降。出生后敲除Dll1损害了肌肉的再生,且显著下调了再生状态下SCs的数目。成年期注射TMX诱导Dll1在肌肉干细胞中特异性敲除,显著下调了稳态下的SCs数目。在单次CTX损伤后,敲除Dll1并不能显著影响再生,但SCs数目显著下调。CTX诱导多次损伤后,Dll1Pax7KO鼠出现严重的再生缺陷且伴随着SCs大量缺失。5.体内外实验显示,敲除Dll1抑制了肌肉干细胞的自我更新,对成肌细胞的增殖无显著影响,但促进了成肌细胞的分化。6.共培养成肌细胞实验证明,野生鼠成肌细胞与Dll1KO成肌细胞共培养后并不能回补Dll1的作用,且Notch信号无差异。综上所述,在肌肉组织中肌纤维源的Dll1对肌肉干细胞的功能无显著影响,而肌肉干细胞特异的Dll1对其功能的维持是必不可少的。Dll1能够通过调控肌肉干细胞的稳态的维持、自我更新和分化来调控肌肉干细胞库。并且在这一调控过程中Dll1以细胞自主的方式调控自身Notch和细胞功能。

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.