A new method of genotyping MDX4CV mice by PCR-RFLP analysis.

INTRODUCTION: The mdx4cv mouse is a common model to study Duchenne muscular dystrophy. The most used methodology to identify the genotype of these mice is Sanger DNA sequencing. METHODS: Here, we provide a simple, cost-effective alternative approach to identify the wild-type, heterozygous, or homozygous/hemizygous genotypes of these mice, using commonly available laboratory equipment and reagents. RESULTS: Our technique exploits a restriction fragment length polymorphism that is generated by the point mutation found in exon 53 of mdx4cv mice. CONCLUSIONS: This technique can benefit laboratories that require complex breeding strategies involving mdx4cv mice. Muscle Nerve 56: 522-524, 2017.

Single-nucleus multiomic analysis of Beckwith-Wiedemann syndrome liver reveals PPARA signaling enrichment and metabolic dysfunction.

Beckwith-Wiedemann Syndrome (BWS) is an epigenetic overgrowth syndrome caused by methylation changes in the human 11p15 chromosomal locus. Patients with BWS exhibit tissue overgrowth, as well as an increased risk of childhood neoplasms in the liver and kidney. To understand the impact of these 11p15 changes, specifically in the liver, we performed single-nucleus RNA sequencing (snRNA-seq) and single-nucleus assay for transposase-accessible chromatin with sequencing (snATAC-seq) to generate paired, cell-type-specific transcriptional and chromatin accessibility profiles of both BWS-liver and nonBWS-liver nontumorous tissue. Our integrated RNA+ATACseq multiomic approach uncovered hepatocyte-specific enrichment and activation of the peroxisome proliferator-activated receptor α (PPARA) - a liver metabolic regulator. To confirm our findings, we utilized a BWS-induced pluripotent stem cell (iPSC) model, where cells were differentiated into hepatocytes. Our data demonstrates the dysregulation of lipid metabolism in BWS-liver, which coincided with observed upregulation of PPARA during hepatocyte differentiation. BWS liver cells exhibited decreased neutral lipids and increased fatty acid ß-oxidation, relative to controls. We also observed increased reactive oxygen species (ROS) byproducts in the form of peroxidated lipids in BWS hepatocytes, which coincided with increased oxidative DNA damage. This study proposes a putative mechanism for overgrowth and cancer predisposition in BWS liver due to perturbed metabolism.

The human DEK oncogene regulates DNA damage response signaling and repair.

The human DEK gene is frequently overexpressed and sometimes amplified in human cancer. Consistent with oncogenic functions, Dek knockout mice are partially resistant to chemically induced papilloma formation. Additionally, DEK knockdown in vitro sensitizes cancer cells to DNA damaging agents and induces cell death via p53-dependent and -independent mechanisms. Here we report that DEK is important for DNA double-strand break repair. DEK depletion in human cancer cell lines and xenografts was sufficient to induce a DNA damage response as assessed by detection of γH2AX and FANCD2. Phosphorylation of H2AX was accompanied by contrasting activation and suppression, respectively, of the ATM and DNA-PK pathways. Similar DNA damage responses were observed in primary Dek knockout mouse embryonic fibroblasts (MEFs), along with increased levels of DNA damage and exaggerated induction of senescence in response to genotoxic stress. Importantly, Dek knockout MEFs exhibited distinct defects in non-homologous end joining (NHEJ) when compared to their wild-type counterparts. Taken together, the data demonstrate new molecular links between DEK and DNA damage response signaling pathways, and suggest that DEK contributes to DNA repair.

Impacts of radiation exposure, hindlimb unloading, and recovery on murine skeletal muscle cell telomere length.

Astronauts are exposed to harsh conditions, including cosmic radiation and microgravity. Spaceflight elongates human telomeres in peripheral blood, which shorten upon return to Earth and approach baseline levels during postflight recovery. Astronauts also encounter muscle atrophy, losing up to 20% loss of muscle mass on spaceflights. Telomere length changes in muscle cells of astronauts remain unexplored. This study investigates telomere alterations in grounded mice experiencing radiation exposure and muscle atrophy, via a hindlimb unloading spaceflight mimicking model. We find telomere lengthening is present in muscle stem cells and in myofiber nuclei, but not in muscle-resident endothelial cells. We further assessed telomere length in the model following hindlimb unloading recovery. We find that telomere length failed to return to baseline values. Our results suggest a role for telomeres in muscle acclimatization, which is relevant for the well-being of astronauts in space, and upon their return to Earth.

CD206+ tendon resident macrophages and their potential crosstalk with fibroblasts and the ECM during tendon growth and maturation.

Resident macrophages exist in a variety of tissues, including tendon, and play context-specific roles in their tissue of residence. In this study, we define the spatiotemporal distribution and phenotypic profile of tendon resident macrophages and their crosstalk with neighboring tendon fibroblasts and the extracellular matrix (ECM) during murine tendon development, growth, and homeostasis. Fluorescent imaging of cryosections revealed that F4/80+ tendon resident macrophages reside adjacent to Col1a1-CFP+ Scx-GFP+ fibroblasts within the tendon fascicle from embryonic development (E15.5) into adulthood (P56). Through flow cytometry and qPCR, we found that these tendon resident macrophages express several well-known macrophage markers, including Adgre1 (F4/80), Mrc1 (CD206), Lyve1, and Folr2, but not Ly-6C, and express the Csf1r-EGFP ("MacGreen") reporter. The proportion of Csf1r-EGFP+ resident macrophages in relation to the total cell number increases markedly during early postnatal growth, while the density of macrophages per mm2 remains constant during this same time frame. Interestingly, proliferation of resident macrophages is higher than adjacent fibroblasts, which likely contributes to this increase in macrophage proportion. The expression profile of tendon resident macrophages also changes with age, with increased pro-inflammatory and anti-inflammatory cytokine expression in P56 compared to P14 macrophages. In addition, the expression profile of limb tendon resident macrophages diverges from that of tail tendon resident macrophages, suggesting differential phenotypes across anatomically and functionally different tendons. As macrophages are known to communicate with adjacent fibroblasts in other tissues, we conducted ligand-receptor analysis and found potential two-way signaling between tendon fibroblasts and resident macrophages. Tendon fibroblasts express high levels of Csf1, which encodes macrophage colony stimulating factor (M-CSF) that acts on the CSF1 receptor (CSF1R) on macrophages. Importantly, Csf1r-expressing resident macrophages preferentially localize to Csf1-expressing fibroblasts, supporting the "nurturing scaffold" model for tendon macrophage patterning. Lastly, we found that tendon resident macrophages express high levels of ECM-related genes, including Mrc1 (mannose receptor), Lyve1 (hyaluronan receptor), Lair1 (type I collagen receptor), Ctss (elastase), and Mmp13 (collagenase), and internalize DQ Collagen in explant cultures. Overall, our study provides insights into the potential roles of tendon resident macrophages in regulating fibroblast phenotype and the ECM during tendon growth.

Specialized Circuitry of Embryonic Stem Cells Promotes Genomic Integrity.

Embryonic stem cells (ESCs) give rise to all cell types of the organism. Given the importance of these cells in this process, ESCs must employ robust mechanisms to protect genomic integrity or risk catastrophic propagation of mutations throughout the organism. Should such an event occur in daughter cells that will eventually contribute to the germline, the overall species health could dramatically decline. This review describes several key mechanisms employed by ESCs that are unique to these cells, in order to maintain their genomic integrity. Additionally, the contributions of cell cycle regulators in modulating ESC differentiation, after DNA damage exposure, are also examined. Where data are available, findings reported in ESCs are extended to include observations described in induced pluripotent stem cells (IPSCs).

Dynamics of skeletal muscle-resident stem cells during myogenesis in fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease in which extraskeletal (heterotopic) bone forms within tissues such as skeletal muscles, often in response to injury. Mutations in the BMP type I receptor ACVR1/ALK2 cause FOP by increasing BMP pathway signaling. In contrast to the growing understanding of the inappropriate formation of bone tissue within the muscle in FOP, much is still unknown about the regenerative capacity of adult diseased muscles. Utilizing an inducible ACVR1R206H knock-in mouse, we found that injured Acvr1R206H/+ skeletal muscle tissue regenerates poorly. We demonstrated that while two resident stem cell populations, muscle stem cells (MuSCs) and fibro/adipogenic progenitors (FAPs), have similar proliferation rates after injury, the differentiation potential of mutant MuSCs is compromised. Although MuSC-specific deletion of the ACVR1R206H mutation does not alter the regenerative potential of skeletal muscles in vivo, Acvr1R206H/+ MuSCs form underdeveloped fibers that fail to fuse in vitro. We further determined that FAPs from Acvr1R206H/+ mice repress the MuSC-mediated formation of Acvr1R206H/+ myotubes in vitro. These results identify a previously unrecognized role for ACVR1R206H in myogenesis in FOP, via improper interaction of tissue-resident stem cells during skeletal muscle regeneration.

Piezo1 regulates the regenerative capacity of skeletal muscles via orchestration of stem cell morphological states.

Muscle stem cells (MuSCs) are essential for tissue homeostasis and regeneration, but the potential contribution of MuSC morphology to in vivo function remains unknown. Here, we demonstrate that quiescent MuSCs are morphologically heterogeneous and exhibit different patterns of cellular protrusions. We classified quiescent MuSCs into three functionally distinct stem cell states: responsive, intermediate, and sensory. We demonstrate that the shift between different stem cell states promotes regeneration and is regulated by the sensing protein Piezo1. Pharmacological activation of Piezo1 is sufficient to prime MuSCs toward more responsive cells. Piezo1 deletion in MuSCs shifts the distribution toward less responsive cells, mimicking the disease phenotype we find in dystrophic muscles. We further demonstrate that Piezo1 reactivation ameliorates the MuSC morphological and regenerative defects of dystrophic muscles. These findings advance our fundamental understanding of how stem cells respond to injury and identify Piezo1 as a key regulator for adjusting stem cell states essential for regeneration.

Telomere length assessments of muscle stem cells in rodent and human skeletal muscle sections.

Measurements of telomere length in skeletal muscle stem cells (MuSCs), a rare cell population within muscles, provide insights into cellular dysfunction in diseased conditions. Here, we describe a protocol (cryosection muscle quantitative fluorescent in situhybridization) using skeletal muscle cryosections for assessments of telomere length in MuSCs, in their native environment. Using a free software, telomere length measurements are assessed on a single-cell level. We also provide methodology to perform data analyses in several ways. For complete details on the use and execution of this protocol, please refer to Tichy et al. (2021).

Persistent NF-κB activation in muscle stem cells induces proliferation-independent telomere shortening.

During the repeated cycles of damage and repair in many muscle disorders, including Duchenne muscular dystrophy (DMD), the muscle stem cell (MuSC) pool becomes less efficient at responding to and repairing damage. The underlying mechanism of such stem cell dysfunction is not fully known. Here, we demonstrate that the distinct early telomere shortening of diseased MuSCs in both mice and young DMD patients is associated with aberrant NF-κB activation. We find that prolonged NF-κB activation in MuSCs in chronic injuries leads to shortened telomeres and Ku80 dysregulation and results in severe skeletal muscle defects. Our studies provide evidence of a role for NF-κB in regulating stem-cell-specific telomere length, independently of cell replication, and could be a congruent mechanism that is applicable to additional tissues and/or diseases characterized by systemic chronic inflammation.

Gli1 Defines a Subset of Fibro-adipogenic Progenitors that Promote Skeletal Muscle Regeneration With Less Fat Accumulation.

Skeletal muscle has remarkable regenerative ability after injury. Mesenchymal fibro-adipogenic progenitors (FAPs) are necessary, active participants during this repair process, but the molecular signatures of these cells and their functional relevance remain largely unexplored. Here, using a lineage tracing mouse model (Gli1-CreER Tomato), we demonstrate that Gli1 marks a small subset of muscle-resident FAPs with elevated Hedgehog (Hh) signaling. Upon notexin muscle injury, these cells preferentially and rapidly expanded within FAPs. Ablation of Gli1+ cells using a DTA mouse model drastically reduced fibroblastic colony-forming unit (CFU-F) colonies generated by muscle cells and impaired muscle repair at 28 days. Pharmacologic manipulation revealed that Gli1+ FAPs rely on Hh signaling to increase the size of regenerating myofiber. Sorted Gli1+ FAPs displayed superior clonogenicity and reduced adipogenic differentiation ability in culture compared to sorted Gli1- FAPs. In a glycerol injury model, Gli1+ FAPs were less likely to give rise to muscle adipocytes compared to other FAPs. Further cell ablation and Hh activator/inhibitor treatments demonstrated their dual actions in enhancing myogenesis and reducing adipogenesis after injury. Examining single-cell RNA-sequencing dataset of FAPs from normal mice indicated that Gli1+ FAPs with increased Hh signaling provide trophic signals to myogenic cells while restrict their own adipogenic differentiation. Collectively, our findings identified a subpopulation of FAPs that play an essential role in skeletal muscle repair. © 2021 American Society for Bone and Mineral Research (ASBMR).

Preservation of genomic integrity in mouse embryonic stem cells.

Embryonic stem (ES) cells and germ cells have the potential to give rise to an entire organism. A common requirement is that both must have very robust mechanisms to preserve the integrity of their genomes. This is particularly true since somatic cells have very high mutation frequencies approaching 10-4 in vivo that would lead to unacceptable levels of fetal lethality and congenital defects. Notably, between 70% and 80% of mutational events monitored at a heterozygous endogenous selectable marker were loss of heterozygosity due to mitotic recombination, a mechanism that affects multiple heterozygous loci between the reporter gene and the site of crossing over. This chapter examines three mechanisms by which mouse embryonic stem cells preserve their genomic integrity. The first entails suppression of mutation and recombination between chromosome homologues by two orders of magnitude when compared with isogenic mouse embryo fibroblasts which had a mutation frequency similar to that seen in adult somatic cells. The second renders mouse ES cells hypersensitive to environmental challenge and eliminates damaged cells from the self-renewing population. Mouse ES cells lack a G1 checkpoint so that cells damaged by exogenous insult such as ionizing radiation do not arrest at the G1/S phase checkpoint but progress into the S phase where the damaged DNA is replicated, the damage exacerbated and the cells driven to apoptosis. The third mechanism examines how mouse ES cells repair double strand DNA breaks. Somatic cells predominantly utilize error prone nonhomologous end joining which, from a teleological perspective, would be disadvantageous for ES cells since it would promote accumulation of mutations. When ES cells were tested for the preferred pathway of double strand DNA break repair, they predominantly utilized the high fidelity homology-mediated repair pathway, thereby minimizing the incurrence of mutations during the repair process. When mouse ES cells are induced to differentiate, the predominant repair pathway switches from homology-mediated repair to nonhomologous end joining that is characteristic of somatic cells.

DNA repair in murine embryonic stem cells and differentiated cells.

Embryonic stem (ES) cells are rapidly proliferating, self-renewing cells that have the capacity to differentiate into all three germ layers to form the embryo proper. Since these cells are critical for embryo formation, they must have robust prophylactic mechanisms to ensure that their genomic integrity is preserved. Indeed, several studies have suggested that ES cells are hypersensitive to DNA damaging agents and readily undergo apoptosis to eliminate damaged cells from the population. Other evidence suggests that DNA damage can cause premature differentiation in these cells. Several laboratories have also begun to investigate the role of DNA repair in the maintenance of ES cell genomic integrity. It does appear that ES cells differ in their capacity to repair damaged DNA compared to differentiated cells. This minireview focuses on repair mechanisms ES cells may use to help preserve genomic integrity and compares available data regarding these mechanisms with those utilized by differentiated cells.

Mechanosensing by the Lamina Protects against Nuclear Rupture, DNA Damage, and Cell-Cycle Arrest.

Whether cell forces or extracellular matrix (ECM) can impact genome integrity is largely unclear. Here, acute perturbations (∼1 h) to actomyosin stress or ECM elasticity cause rapid and reversible changes in lamin-A, DNA damage, and cell cycle. The findings are especially relevant to organs such as the heart because DNA damage permanently arrests cardiomyocyte proliferation shortly after birth and thereby eliminates regeneration after injury including heart attack. Embryonic hearts, cardiac-differentiated iPS cells (induced pluripotent stem cells), and various nonmuscle cell types all show that actomyosin-driven nuclear rupture causes cytoplasmic mis-localization of DNA repair factors and excess DNA damage. Binucleation and micronuclei increase as telomeres shorten, which all favor cell-cycle arrest. Deficiencies in lamin-A and repair factors exacerbate these effects, but lamin-A-associated defects are rescued by repair factor overexpression and also by contractility modulators in clinical trials. Contractile cells on stiff ECM normally exhibit low phosphorylation and slow degradation of lamin-A by matrix-metalloprotease-2 (MMP2), and inhibition of this lamin-A turnover and also actomyosin contractility are seen to minimize DNA damage. Lamin-A is thus stress stabilized to mechano-protect the genome.

Human Skeletal Stem Cells: The Markers Provide Some Clues in the Hunt for Hidden Treasure.

Little is known about skeletal stem cell populations in vivo. Recently in Cell, Chan et al. (2018) identified a human skeletal stem cell population that can be isolated from multiple human bone locations and is capable of self-renewal and differentiation into bone, cartilage, and stroma, but not fat.

A robust Pax7EGFP mouse that enables the visualization of dynamic behaviors of muscle stem cells.

BACKGROUND: Pax7 is a transcription factor involved in the specification and maintenance of muscle stem cells (MuSCs). Upon injury, MuSCs leave their quiescent state, downregulate Pax7 and differentiate, contributing to skeletal muscle regeneration. In the majority of regeneration studies, MuSCs are isolated by fluorescence-activated sorting (FACS), based on cell surface markers. It is known that MuSCs are a heterogeneous population and only a small percentage of isolated cells are true stem cells that are able to self-renew. A strong Pax7 reporter line would be valuable to study the in vivo behavior of Pax7-expressing stem cells. METHODS: We generated and characterized the muscle properties of a new transgenic Pax7EGFP mouse. Utilizing traditional immunofluorescence assays, we analyzed whole embryos and muscle sections by fluorescence microscopy, in addition to whole skeletal muscles by 2-photon microscopy, to detect the specificity of EGFP expression. Skeletal muscles from Pax7EGFP mice were also evaluated in steady state and under injury conditions. Finally, MuSCs-derived from Pax7EGFP and control mice were sorted and analyzed by FACS and their myogenic activity was comparatively examined. RESULTS: Our studies provide a new Pax7 reporter line with robust EGFP expression, detectable by both flow cytometry and fluorescence microscopy. Pax7EGFP-derived MuSCs have identical properties to that of wild-type MuSCs, both in vitro and in vivo, excluding any positional effect due to the transgene insertion. Furthermore, we demonstrated high specificity of EGFP to label MuSCs in a temporal manner that recapitulates the reported Pax7 expression pattern. Interestingly, immunofluorescence analysis showed that the robust expression of EGFP marks cells in the satellite cell position of adult muscles in fixed and live tissues. CONCLUSIONS: This mouse could be an invaluable tool for the study of a variety of questions related to MuSC biology, including but not limited to population heterogeneity, polarity, aging, regeneration, and motility, either by itself or in combination with mice harboring additional genetic alterations.

Single Stem Cell Imaging and Analysis Reveals Telomere Length Differences in Diseased Human and Mouse Skeletal Muscles.

Muscle stem cells (MuSCs) contribute to muscle regeneration following injury. In many muscle disorders, the repeated cycles of damage and repair lead to stem cell dysfunction. While telomere attrition may contribute to aberrant stem cell functions, methods to accurately measure telomere length in stem cells from skeletal muscles have not been demonstrated. Here, we have optimized and validated such a method, named MuQ-FISH, for analyzing telomere length in MuSCs from either mice or humans. Our analysis showed no differences in telomere length between young and aged MuSCs from uninjured wild-type mice, but MuSCs isolated from young dystrophic mice exhibited significantly shortened telomeres. In corroboration, we demonstrated that telomere attrition is present in human dystrophic MuSCs, which underscores its importance in diseased regenerative failure. The robust technique described herein provides analysis at a single-cell resolution and may be utilized for other cell types, especially rare populations of cells.

Cardiomyocyte-Specific Telomere Shortening is a Distinct Signature of Heart Failure in Humans.

BACKGROUND: Telomere defects are thought to play a role in cardiomyopathies, but the specific cell type affected by the disease in human hearts is not yet identified. The aim of this study was to systematically evaluate the cell type specificity of telomere shortening in patients with heart failure in relation to their cardiac disease, age, and sex. METHODS AND RESULTS: We studied cardiac tissues from patients with heart failure by utilizing telomere quantitative fluorescence in situ hybridization, a highly sensitive method with single-cell resolution. In this study, total of 63 human left ventricular samples, including 37 diseased and 26 nonfailing donor hearts, were stained for telomeres in combination with cardiomyocyte- or α-smooth muscle cell-specific markers, cardiac troponin T, and smooth muscle actin, respectively, and assessed for telomere length. Patients with heart failure demonstrate shorter cardiomyocyte telomeres compared with nonfailing donors, which is specific only to cardiomyocytes within diseased human hearts and is associated with cardiomyocyte DNA damage. Our data further reveal that hypertrophic hearts with reduced ejection fraction exhibit the shortest telomeres. In contrast to other reported cell types, no difference in cardiomyocyte telomere length is evident with age. However, under the disease state, telomere attrition manifests in both young and older patients with cardiac hypertrophy. Finally, we demonstrate that cardiomyocyte-telomere length is better sustained in women than men under diseased conditions. CONCLUSIONS: This study provides the first evidence of cardiomyocyte-specific telomere shortening in heart failure.

Loss of NAD Homeostasis Leads to Progressive and Reversible Degeneration of Skeletal Muscle.

NAD is an obligate co-factor for the catabolism of metabolic fuels in all cell types. However, the availability of NAD in several tissues can become limited during genotoxic stress and the course of natural aging. The point at which NAD restriction imposes functional limitations on tissue physiology remains unknown. We examined this question in murine skeletal muscle by specifically depleting Nampt, an essential enzyme in the NAD salvage pathway. Knockout mice exhibited a dramatic 85% decline in intramuscular NAD content, accompanied by fiber degeneration and progressive loss of both muscle strength and treadmill endurance. Administration of the NAD precursor nicotinamide riboside rapidly ameliorated functional deficits and restored muscle mass despite having only a modest effect on the intramuscular NAD pool. Additionally, lifelong overexpression of Nampt preserved muscle NAD levels and exercise capacity in aged mice, supporting a critical role for tissue-autonomous NAD homeostasis in maintaining muscle mass and function.

Mouse embryonic stem cells undergo charontosis, a novel programmed cell death pathway dependent upon cathepsins, p53, and EndoG, in response to etoposide treatment.

Embryonic stem cells (ESCs) are hypersensitive to many DNA damaging agents and can rapidly undergo cell death or cell differentiation following exposure. Treatment of mouse ESCs (mESCs) with etoposide (ETO), a topoisomerase II poison, followed by a recovery period resulted in massive cell death with characteristics of a programmed cell death pathway (PCD). While cell death was both caspase- and necroptosis-independent, it was partially dependent on the activity of lysosomal proteases. A role for autophagy in the cell death process was eliminated, suggesting that ETO induces a novel PCD pathway in mESCs. Inhibition of p53 either as a transcription factor by pifithrin α or in its mitochondrial role by pifithrin µ significantly reduced ESC death levels. Finally, EndoG was newly identified as a protease participating in the DNA fragmentation observed during ETO-induced PCD. We coined the term charontosis after Charon, the ferryman of the dead in Greek mythology, to refer to the PCD signaling events induced by ETO in mESCs.