Aging disrupts gene expression timing during muscle regeneration.

Skeletal muscle function and regenerative capacity decline during aging, yet factors driving these changes are incompletely understood. Muscle regeneration requires temporally coordinated transcriptional programs to drive myogenic stem cells to activate, proliferate, fuse to form myofibers, and to mature as myonuclei, restoring muscle function after injury. We assessed global changes in myogenic transcription programs distinguishing muscle regeneration in aged mice from young mice by comparing pseudotime trajectories from single-nucleus RNA sequencing of myogenic nuclei. Aging-specific differences in coordinating myogenic transcription programs necessary for restoring muscle function occur following muscle injury, likely contributing to compromised regeneration in aged mice. Differences in pseudotime alignment of myogenic nuclei when comparing aged with young mice via dynamic time warping revealed pseudotemporal differences becoming progressively more severe as regeneration proceeds. Disruptions in timing of myogenic gene expression programs may contribute to incomplete skeletal muscle regeneration and declines in muscle function as organisms age.

Photo-expansion microscopy enables super-resolution imaging of cells embedded in 3D hydrogels.

Hydrogels are extensively used as tunable, biomimetic three-dimensional cell culture matrices, but optically deep, high-resolution images are often difficult to obtain, limiting nanoscale quantification of cell-matrix interactions and outside-in signalling. Here we present photopolymerized hydrogels for expansion microscopy that enable optical clearance and tunable ×4.6-6.7 homogeneous expansion of not only monolayer cell cultures and tissue sections, but cells embedded within hydrogels. The photopolymerized hydrogels for expansion microscopy formulation relies on a rapid photoinitiated thiol/acrylate mixed-mode polymerization that is not inhibited by oxygen and decouples monomer diffusion from polymerization, which is particularly beneficial when expanding cells embedded within hydrogels. Using this technology, we visualize human mesenchymal stem cells and their interactions with nascently deposited proteins at <120 nm resolution when cultured in proteolytically degradable synthetic polyethylene glycol hydrogels. Results support the notion that focal adhesion maturation requires cellular fibronectin deposition; nuclear deformation precedes cellular spreading; and human mesenchymal stem cells display cell-surface metalloproteinases for matrix remodelling.

The regenerating skeletal muscle niche drives satellite cell return to quiescence.

Skeletal muscle stem cells, or satellite cells (SCs), are essential to regenerate and maintain muscle. Quiescent SCs reside in an asymmetric niche between the basal lamina and myofiber membrane. To repair muscle, SCs activate, proliferate, and differentiate, fusing to repair myofibers or reacquiring quiescence to replenish the SC niche. Little is known about when SCs reacquire quiescence during regeneration or the cellular processes that direct SC fate decisions. We find that most SCs reacquire quiescence 5-10 days after muscle injury, following differentiation and fusion of most cells to regenerate myofibers. Single-cell sequencing of myogenic cells in regenerating muscle identifies SCs reacquiring quiescence and reveals that noncell autonomous signaling networks influence SC fate decisions during regeneration. SC transplantation experiments confirm that the regenerating environment influences SC fate. We define a window for SC repopulation of the niche, emphasizing the temporal contribution of the regenerative muscle environment on SC fate.

RNA-binding proteins direct myogenic cell fate decisions.

RNA-binding proteins (RBPs), essential for skeletal muscle regeneration, cause muscle degeneration and neuromuscular disease when mutated. Why mutations in these ubiquitously expressed RBPs orchestrate complex tissue regeneration and direct cell fate decisions in skeletal muscle remains poorly understood. Single-cell RNA-sequencing of regenerating Mus musculus skeletal muscle reveals that RBP expression, including the expression of many neuromuscular disease-associated RBPs, is temporally regulated in skeletal muscle stem cells and correlates with specific stages of myogenic differentiation. By combining machine learning with RBP engagement scoring, we discovered that the neuromuscular disease-associated RBP Hnrnpa2b1 is a differentiation-specifying regulator of myogenesis that controls myogenic cell fate transitions during terminal differentiation in mice. The timing of RBP expression specifies cell fate transitions by providing post-transcriptional regulation of messenger RNAs that coordinate stem cell fate decisions during tissue regeneration.

Injury-mediated stiffening persistently activates muscle stem cells through YAP and TAZ mechanotransduction.

The skeletal muscle microenvironment transiently remodels and stiffens after exercise and injury, as muscle ages, and in myopathic muscle; however, how these changes in stiffness affect resident muscle stem cells (MuSCs) remains understudied. Following muscle injury, muscle stiffness remained elevated after morphological regeneration was complete, accompanied by activated and proliferative MuSCs. To isolate the role of stiffness on MuSC behavior and determine the underlying mechanotransduction pathways, we cultured MuSCs on strain-promoted azide-alkyne cycloaddition hydrogels capable of in situ stiffening by secondary photocrosslinking of excess cyclooctynes. Using pre- to post-injury stiffness hydrogels, we found that elevated stiffness enhances migration and MuSC proliferation by localizing yes-associated protein 1 (YAP) and WW domain-containing transcription regulator 1 (WWTR1; TAZ) to the nucleus. Ablating YAP and TAZ in vivo promotes MuSC quiescence in postinjury muscle and prevents myofiber hypertrophy, demonstrating that persistent exposure to elevated stiffness activates mechanotransduction signaling maintaining activated and proliferating MuSCs.

Cosmc is required for T cell persistence in the periphery.

T lymphocytes, a key arm of adaptive immunity, are known to dynamically regulate O-glycosylation during T cell maturation and when responding to stimuli; however, the direct role of O-glycans in T cell maturation remains largely unknown. Using a conditional knockout of the gene (C1GalT1C1 or Cosmc) encoding the specific chaperone Cosmc, we generated mice whose T cells lack extended O-glycans (T cell conditional Cosmc knock out or TCKO mice) and homogeneously express the truncated Tn antigen. Loss of Cosmc is highly deleterious to T cell persistence, with near-complete elimination of Cosmc-null T cells from spleen and lymph nodes. Total T cell counts are 20% of wild type (WT), among which only 5% express the truncated glycans, with the remaining 95% consisting of escapers from Cre-mediated recombination. TCKO thymocytes were able to complete thymic maturation but failed to populate the secondary lymphoid organs both natively and upon adoptive transfer to WT recipients. Our results demonstrate that extended O-glycosylation is required for the establishment and maintenance of the peripheral T cell population.

Myo-granules Connect Physiology and Pathophysiology.

A hallmark of many neuromuscular diseases including Alzheimer disease, inclusion body myositis, amyotrophic lateral sclerosis, frontotemporal lobar dementia, and ocular pharyngeal muscular dystrophy is large cytoplasmic aggregates containing the RNA-binding protein, TDP-43. Despite acceptance that cytoplasmic TDP-43 aggregation is pathological, cytoplasmic TDP-43 assemblies form in healthy regenerating muscle. These recently discovered ribonucleoprotein assemblies, termed myo-granules, form in healthy muscle following injury and are readily cleared as the myofibers mature. The formation and dissolution of myo-granules during normal muscle regeneration suggests that these amyloid-like oligomers may be functional and that perturbations in myo-granule kinetics or composition may promote pathological aggregation.

Muscling in on the Awesome Proliferative Power of the Terrible Teratoma.

In this issue of Cell Stem Cell, Chan et al. (2018) report that in vivo differentiation of pluripotent stem cells in induced teratomas produces functional embryonic-like muscle stem cells. These purified muscle stem cells engraft with high efficiency and regenerate serially injured muscle.

Non-equivalence of nuclear import among nuclei in multinucleated skeletal muscle cells.

Skeletal muscle is primarily composed of large myofibers containing thousands of post-mitotic nuclei distributed throughout a common cytoplasm. Protein production and localization in specialized myofiber regions is crucial for muscle function. Myonuclei differ in transcriptional activity and protein accumulation, but how these differences among nuclei sharing a cytoplasm are achieved is unknown. Regulated nuclear import of proteins is one potential mechanism for regulating transcription spatially and temporally in individual myonuclei. The best-characterized nuclear localization signal (NLS) in proteins is the classical NLS (cNLS), but many other NLS motifs exist. We examined cNLS and non-cNLS reporter protein import using multinucleated muscle cells generated in vitro, revealing that cNLS and non-cNLS nuclear import differs among nuclei in the same cell. Investigation of cNLS nuclear import rates in isolated myofibers ex vivo confirmed differences in nuclear import rates among myonuclei. Analyzing nuclear import throughout myogenesis revealed that cNLS and non-cNLS import varies during differentiation. Taken together, our results suggest that both spatial and temporal regulation of nuclear import pathways are important in muscle cell differentiation and protein regionalization in myofibers.

The RNA-binding protein, ZC3H14, is required for proper poly(A) tail length control, expression of synaptic proteins, and brain function in mice.

A number of mutations in genes that encode ubiquitously expressed RNA-binding proteins cause tissue specific disease. Many of these diseases are neurological in nature revealing critical roles for this class of proteins in the brain. We recently identified mutations in a gene that encodes a ubiquitously expressed polyadenosine RNA-binding protein, ZC3H14 (Zinc finger CysCysCysHis domain-containing protein 14), that cause a nonsyndromic, autosomal recessive form of intellectual disability. This finding reveals the molecular basis for disease and provides evidence that ZC3H14 is essential for proper brain function. To investigate the role of ZC3H14 in the mammalian brain, we generated a mouse in which the first common exon of the ZC3H14 gene, exon 13 is removed (Zc3h14Δex13/Δex13) leading to a truncated ZC3H14 protein. We report here that, as in the patients, Zc3h14 is not essential in mice. Utilizing these Zc3h14Δex13/Δex13mice, we provide the first in vivo functional characterization of ZC3H14 as a regulator of RNA poly(A) tail length. The Zc3h14Δex13/Δex13 mice show enlarged lateral ventricles in the brain as well as impaired working memory. Proteomic analysis comparing the hippocampi of Zc3h14+/+ and Zc3h14Δex13/Δex13 mice reveals dysregulation of several pathways that are important for proper brain function and thus sheds light onto which pathways are most affected by the loss of ZC3H14. Among the proteins increased in the hippocampi of Zc3h14Δex13/Δex13 mice compared to control are key synaptic proteins including CaMK2a. This newly generated mouse serves as a tool to study the function of ZC3H14 in vivo.

Biochemical isolation of myonuclei employed to define changes to the myonuclear proteome that occur with aging.

Skeletal muscle aging is accompanied by loss of muscle mass and strength. Examining changes in myonuclear proteins with age would provide insight into molecular processes which regulate these profound changes in muscle physiology. However, muscle tissue is highly adapted for contraction and thus comprised largely of contractile proteins making the nuclear proteins difficult to identify from whole muscle samples. By developing a method to purify myonuclei from whole skeletal muscle, we were able to collect myonuclei for analysis by flow cytometry, biochemistry, and mass spectrometry. Nuclear purification dramatically increased the number and intensity of nuclear proteins detected by mass spectrometry compared to whole tissue. We exploited this increased proteomic depth to investigate age-related changes to the myonuclear proteome. Nuclear levels of 54 of 779 identified proteins (7%) changed significantly with age; these proteins were primarily involved in chromatin maintenance and RNA processing. To determine whether the changes we detected were specific to myonuclei or were common to nuclei of excitatory tissues, we compared aging in myonuclei to aging in brain nuclei. Although several of the same processes were affected by aging in both brain and muscle nuclei, the specific proteins involved in these alterations differed between the two tissues. Isolating myonuclei allowed a deeper view into the myonuclear proteome than previously possible facilitating identification of novel age-related changes in skeletal muscle. Our technique will enable future studies into a heretofore underrepresented compartment of skeletal muscle.

Biochemical Isolation of Myonuclei from Mouse Skeletal Muscle Tissue.

Skeletal muscle provides the contractile force necessary for movement, swallowing, and breathing and, consequently, is necessary for survival. Skeletal muscle cells are unique in that they are extremely large cells containing thousands of nuclei. These nuclei must all work in concert to maintain skeletal muscle function and thereby maintain life. The nucleus is a major site of signaling integration and gene expression regulation. However, examining nuclear processes in skeletal muscle can be difficult because myonuclei are challenging to isolate. We optimized a protocol to purify myonuclei from whole muscle tissue using ultracentrifugation over a discontinuous sucrose gradient to separate the nuclear fraction. We used these purified nuclei for downstream applications including flow cytometry and mass spectrometry. We used this method to compare the myonuclear proteome of young and old mouse hindlimb muscles (Cutler et al., 2017). This protocol may be applied to isolating myonuclei for a variety of downstream analyses such as flow cytometry, microscopy, Western blot, and proteomics.

Karyopherin Alpha 1 Regulates Satellite Cell Proliferation and Survival by Modulating Nuclear Import.

Satellite cells are stem cells with an essential role in skeletal muscle repair. Precise regulation of gene expression is critical for proper satellite cell quiescence, proliferation, differentiation and self-renewal. Nuclear proteins required for gene expression are dependent on the nucleocytoplasmic transport machinery to access to nucleus, however little is known about regulation of nuclear transport in satellite cells. The best characterized nuclear import pathway is classical nuclear import which depends on a classical nuclear localization signal (cNLS) in a cargo protein and the heterodimeric import receptors, karyopherin alpha (KPNA) and beta (KPNB). Multiple KPNA1 paralogs exist and can differ in importing specific cNLS proteins required for cell differentiation and function. We show that transcripts for six Kpna paralogs underwent distinct changes in mouse satellite cells during muscle regeneration accompanied by changes in cNLS proteins in nuclei. Depletion of KPNA1, the most dramatically altered KPNA, caused satellite cells in uninjured muscle to prematurely activate, proliferate and undergo apoptosis leading to satellite cell exhaustion with age. Increased proliferation of satellite cells led to enhanced muscle regeneration at early stages of regeneration. In addition, we observed impaired nuclear localization of two key KPNA1 cargo proteins: p27, a cyclin-dependent kinase inhibitor associated with cell cycle control and lymphoid enhancer factor 1, a critical cotranscription factor for ß-catenin. These results indicate that regulated nuclear import of proteins by KPNA1 is critical for satellite cell proliferation and survival and establish classical nuclear import as a novel regulatory mechanism for controlling satellite cell fate. Stem Cells 2016;34:2784-2797.

Corneal myofibroblast generation from bone marrow-derived cells.

The purpose of this study was to determine whether bone marrow-derived cells can differentiate into myofibroblasts, as defined by alpha-smooth muscle actin (SMA) expression, that arise in the corneal stroma after irregular phototherapeutic keratectomy and whose presence within the cornea is associated with corneal stromal haze. C57BL/6J-GFP chimeric mice were generated through bone marrow transplantation from donor mice that expressed enhanced green fluorescent protein (GFP) in a high proportion of their bone marrow-derived cells. Twenty-four GFP chimeric mice underwent haze-generating corneal epithelial scrape followed by irregular phototherapeutic keratectomy (PTK) with an excimer laser in one eye. Mice were euthanized at 2 weeks or 4 weeks after PTK and the treated and control contralateral eyes were removed and cryo-preserved for sectioning for immunocytochemistry. Double immunocytochemistry for GFP and myofibroblast marker alpha-smooth muscle actin (SMA) were performed and the number of SMA+GFP+, SMA+GFP-, SMA-GFP+ and SMA-GFP- cells, as well as the number of DAPI+ cell nuclei, per 400x field of stroma was determined in the central, mid-peripheral and peri-limbal cornea. In this mouse model, there were no SMA+ cells and only a few GFP+ cells detected in unwounded control corneas. No SMA+ cells were detected in the stroma at two weeks after irregular PTK, even though there were numerous GFP+ cells present. At 4 weeks after irregular PTK, all corneas developed mild to moderately severe corneal haze. In each of the three regions of the corneas examined, there were on average more than 9x more SMA+GFP+ than SMA+GFP- myofibroblasts. This difference was significant (p < 0.01). There were significantly more (p < 0.01) SMA-GFP+ cells, which likely include inflammatory cells, than SMA+GFP+ or SMA+GFP- cells, although SMA-GFP- cells represent the largest population of cells in the corneas. In this mouse model, the majority of myofibroblasts developed from bone marrow-derived cells. It is possible that all myofibroblasts in these animals developed from bone marrow-derived cells since mouse chimeras produced using this method had only 60-95% of bone marrow-derived cells that were GFP+ and it is not possible to achieve 100% chimerization. This model, therefore, cannot exclude the possibility of myofibroblasts also developed from keratocytes and/or corneal fibroblasts.