Publications by authors named "Foteini Mourkioti"

27 Publications

  • Page 1 of 1

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

Cell Rep 2021 May;35(6):109098

Department of Orthopaedic Surgery, Perelman School of Medicine, The University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Cell and Developmental Biology, Perelman School of Medicine, The University of Pennsylvania, Philadelphia, PA 19104, USA; Institute of Regenerative Medicine, Musculoskeletal Regeneration Program, Perelman School of Medicine, The University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2021.109098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8183356PMC
May 2021

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

J Bone Miner Res 2021 Feb 2. Epub 2021 Feb 2.

Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA.

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).
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http://dx.doi.org/10.1002/jbmr.4265DOI Listing
February 2021

Pre-innervated tissue-engineered muscle promotes a pro-regenerative microenvironment following volumetric muscle loss.

Commun Biol 2020 06 25;3(1):330. Epub 2020 Jun 25.

Center for Brain Injury & Repair, Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Volumetric muscle loss (VML) is the traumatic or surgical loss of skeletal muscle beyond the inherent regenerative capacity of the body, generally leading to severe functional deficit. Formation of appropriate somato-motor innervations remains one of the biggest challenges for both autologous grafts as well as tissue-engineered muscle constructs. We aim to address this challenge by developing pre-innervated tissue-engineered muscle comprised of long aligned networks of spinal motor neurons and skeletal myocytes on aligned nanofibrous scaffolds. Motor neurons led to enhanced differentiation and maturation of skeletal myocytes in vitro. These pre-innervated tissue-engineered muscle constructs when implanted in a rat VML model significantly increased satellite cell density, neuromuscular junction maintenance, graft revascularization, and muscle volume over three weeks as compared to myocyte-only constructs and nanofiber scaffolds alone. These pro-regenerative effects may enhance functional neuromuscular regeneration following VML, thereby improving the levels of functional recovery following these devastating injuries.
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http://dx.doi.org/10.1038/s42003-020-1056-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7316777PMC
June 2020

Innervation: the missing link for biofabricated tissues and organs.

NPJ Regen Med 2020 5;5:11. Epub 2020 Jun 5.

Center for Brain Injury & Repair, Department of Neurosurgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA USA.

Innervation plays a pivotal role as a driver of tissue and organ development as well as a means for their functional control and modulation. Therefore, innervation should be carefully considered throughout the process of biofabrication of engineered tissues and organs. Unfortunately, innervation has generally been overlooked in most non-neural tissue engineering applications, in part due to the intrinsic complexity of building organs containing heterogeneous native cell types and structures. To achieve proper innervation of engineered tissues and organs, specific host axon populations typically need to be precisely driven to appropriate location(s) within the construct, often over long distances. As such, neural tissue engineering and/or axon guidance strategies should be a necessary adjunct to most organogenesis endeavors across multiple tissue and organ systems. To address this challenge, our team is actively building axon-based "living scaffolds" that may physically wire in during organ development in bioreactors and/or serve as a substrate to effectively drive targeted long-distance growth and integration of host axons after implantation. This article reviews the neuroanatomy and the role of innervation in the functional regulation of cardiac, skeletal, and smooth muscle tissue and highlights potential strategies to promote innervation of biofabricated engineered muscles, as well as the use of "living scaffolds" in this endeavor for both in vitro and in vivo applications. We assert that innervation should be included as a necessary component for tissue and organ biofabrication, and that strategies to orchestrate host axonal integration are advantageous to ensure proper function, tolerance, assimilation, and bio-regulation with the recipient post-implant.
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http://dx.doi.org/10.1038/s41536-020-0096-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275031PMC
June 2020

Elevated BMP and Mechanical Signaling Through YAP1/RhoA Poises FOP Mesenchymal Progenitors for Osteogenesis.

J Bone Miner Res 2019 10 19;34(10):1894-1909. Epub 2019 Aug 19.

Department of Orthopaedic Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by the formation of extraskeletal bone, or heterotopic ossification (HO), in soft connective tissues such as skeletal muscle. All familial and sporadic cases with a classic clinical presentation of FOP carry a gain-of-function mutation (R206H; c.617 G > A) in ACVR1, a cell surface receptor that mediates bone morphogenetic protein (BMP) signaling. The BMP signaling pathway is recognized for its chondro/osteogenic-induction potential, and HO in FOP patients forms ectopic but qualitatively normal endochondral bone tissue through misdirected cell fate decisions by tissue-resident mesenchymal stem cells. In addition to biochemical ligand-receptor signaling, mechanical cues from the physical environment are transduced to activate intracellular signaling, a process known as mechanotransduction, and can influence cell fates. Utilizing an established mesenchymal stem cell model of mouse embryonic fibroblasts (MEFs) from the Acvr1 mouse model that mimics the human disease, we demonstrated that activation of the mechanotransductive effectors Rho/ROCK and YAP1 are increased in Acvr1 cells. We show that on softer substrates, a condition associated with low mechanical signaling, the morphology of Acvr1 cells is similar to the morphology of control Acvr1 cells on stiffer substrates, a condition that activates mechanotransduction. We further determined that Acvr1 cells are poised for osteogenic differentiation, expressing increased levels of chondro/osteogenic markers compared with Acvr1 cells. We also identified increased YAP1 nuclear localization in Acvr1 cells, which can be rescued by either BMP inhibition or Rho antagonism. Our results establish RhoA and YAP1 signaling as modulators of mechanotransduction in FOP and suggest that aberrant mechanical signals, combined with and as a result of the increased BMP pathway signaling through mutant ACVR1, lead to misinterpretation of the cellular microenvironment and a heightened sensitivity to mechanical stimuli that promotes commitment of Acvr1 progenitor cells to chondro/osteogenic lineages.
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http://dx.doi.org/10.1002/jbmr.3760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7209824PMC
October 2019

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

Dev Cell 2019 06 16;49(6):920-935.e5. Epub 2019 May 16.

Molecular & Cell Biophysics Laboratory, University of Pennsylvania, Philadelphia, PA 19104, USA; Orthopaedic Surgery and Cell & Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Penn Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.devcel.2019.04.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6581604PMC
June 2019

ACVR1 FOP mutation alters mechanosensing and tissue stiffness during heterotopic ossification.

Mol Biol Cell 2019 01 31;30(1):17-29. Epub 2018 Oct 31.

Department of Orthopaedic Surgery, University of Pennsylvania, Philadelphia, PA 19104.

An activating bone morphogenetic proteins (BMP) type I receptor ACVR1 (ACVR1) mutation enhances BMP pathway signaling and causes the rare genetic disorder of heterotopic (extraskeletal) bone formation fibrodysplasia ossificans progressiva. Heterotopic ossification frequently occurs following injury as cells aberrantly differentiate during tissue repair. Biomechanical signals from the tissue microenvironment and cellular responses to these physical cues, such as stiffness and rigidity, are important determinants of cell differentiation and are modulated by BMP signaling. We used an Acvr1 mouse model of injury-induced heterotopic ossification to examine the fibroproliferative tissue preceding heterotopic bone and identified pathologic stiffening at this stage of repair. In response to microenvironment stiffness, in vitro assays showed that Acvr1 cells inappropriately sense their environment, responding to soft substrates with a spread morphology similar to wild-type cells on stiff substrates and to cells undergoing osteoblastogenesis. Increased activation of RhoA and its downstream effectors demonstrated increased mechanosignaling. Nuclear localization of the pro-osteoblastic factor RUNX2 on soft and stiff substrates suggests a predisposition to this cell fate. Our data support that increased BMP signaling in Acvr1 cells alters the tissue microenvironment and results in misinterpretation of the tissue microenvironment through altered sensitivity to mechanical stimuli that lowers the threshold for commitment to chondro/osteogenic lineages.
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http://dx.doi.org/10.1091/mbc.E18-05-0311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6337906PMC
January 2019

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

Cell Stem Cell 2018 10;23(4):462-463

Department of Orthopaedic Surgery, Perelman School of Medicine, The University of Pennsylvania, Philadelphia, PA, USA; Department of Cell and Developmental Biology, Perelman School of Medicine, The University of Pennsylvania, Philadelphia, PA, USA; Penn Institute for Regenerative Medicine, Musculoskeletal Regeneration Program, Perelman School of Medicine, The University of Pennsylvania, Philadelphia, PA, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.stem.2018.09.006DOI Listing
October 2018

Engineered DNA plasmid reduces immunity to dystrophin while improving muscle force in a model of gene therapy of Duchenne dystrophy.

Proc Natl Acad Sci U S A 2018 09 4;115(39):E9182-E9191. Epub 2018 Sep 4.

Department of Neurology, Stanford University, Stanford, CA 94304;

In gene therapy for Duchenne muscular dystrophy there are two potential immunological obstacles. An individual with Duchenne muscular dystrophy has a genetic mutation in dystrophin, and therefore the wild-type protein is "foreign," and thus potentially immunogenic. The adeno-associated virus serotype-6 (AAV6) vector for delivery of dystrophin is a viral-derived vector with its own inherent immunogenicity. We have developed a technology where an engineered plasmid DNA is delivered to reduce autoimmunity. We have taken this approach into humans, tolerizing to myelin proteins in multiple sclerosis and to proinsulin in type 1 diabetes. Here, we extend this technology to a model of gene therapy to reduce the immunogenicity of the AAV vector and of the wild-type protein product that is missing in the genetic disease. Following gene therapy with systemic administration of recombinant AAV6-microdystrophin to mdx/mTR mice, we demonstrated the development of antibodies targeting dystrophin and AAV6 capsid in control mice. Treatment with the engineered DNA construct encoding microdystrophin markedly reduced antibody responses to dystrophin and to AAV6. Muscle force in the treated mice was also improved compared with control mice. These data highlight the potential benefits of administration of an engineered DNA plasmid encoding the delivered protein to overcome critical barriers in gene therapy to achieve optimal functional gene expression.
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http://dx.doi.org/10.1073/pnas.1808648115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166850PMC
September 2018

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

Skelet Muscle 2018 08 24;8(1):27. Epub 2018 Aug 24.

Department of Orthopaedic Surgery, Perelman School of Medicine, The University of Pennsylvania, Philadelphia, PA, USA.

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.
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http://dx.doi.org/10.1186/s13395-018-0169-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6107960PMC
August 2018

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

Stem Cell Reports 2017 10 7;9(4):1328-1341. Epub 2017 Sep 7.

Department of Orthopaedic Surgery, Perelman School of Medicine, The University of Pennsylvania, 112A Stemmler Hall, 3450 Hamilton Walk, Philadelphia, PA 19104-6081, USA; Department of Cell and Developmental Biology, Perelman School of Medicine, The University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.stemcr.2017.08.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5639167PMC
October 2017

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

J Am Heart Assoc 2017 Sep 7;6(9). Epub 2017 Sep 7.

Department of Orthopaedic Surgery, University of Connecticut, Storrs, CT

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.
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http://dx.doi.org/10.1161/JAHA.116.005086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5634248PMC
September 2017

Evaluation of telomere length in human cardiac tissues using cardiac quantitative FISH.

Nat Protoc 2017 Sep 17;12(9):1855-1870. Epub 2017 Aug 17.

Department of Orthopaedic Surgery, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Telomere length has been correlated with various diseases, including cardiovascular disease and cancer. The use of currently available telomere-length measurement techniques is often restricted by the requirement of a large amount of cells (Southern-based techniques) or the lack of information on individual cells or telomeres (PCR-based methods). Although several methods have been used to measure telomere length in tissues as a whole, the assessment of cell-type-specific telomere length provides valuable information on individual cell types. The development of fluorescence in situ hybridization (FISH) technologies enables the quantification of telomeres in individual chromosomes, but the use of these methods is dependent on the availability of isolated cells, which prevents their use with fixed archival samples. Here we describe an optimized quantitative FISH (Q-FISH) protocol for measuring telomere length that bypasses the previous limitations by avoiding contributions from undesired cell types. We have used this protocol on small paraffin-embedded cardiac-tissue samples. This protocol describes step-by-step procedures for tissue preparation, permeabilization, cardiac-tissue pretreatment and hybridization with a Cy3-labeled telomeric repeat complementing (CCCTAA) peptide nucleic acid (PNA) probe coupled with cardiac-specific antibody staining. We also describe how to quantify telomere length by means of the fluorescence intensity and area of each telomere within individual nuclei. This protocol provides comparative cell-type-specific telomere-length measurements in relatively small human cardiac samples and offers an attractive technique to test hypotheses implicating telomere length in various cardiac pathologies. The current protocol (from tissue collection to image procurement) takes ∼28 h along with three overnight incubations. We anticipate that the protocol could be easily adapted for use on different tissue types.
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http://dx.doi.org/10.1038/nprot.2017.082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7716275PMC
September 2017

Long telomeres protect against age-dependent cardiac disease caused by NOTCH1 haploinsufficiency.

J Clin Invest 2017 May 27;127(5):1683-1688. Epub 2017 Mar 27.

Diseases caused by gene haploinsufficiency in humans commonly lack a phenotype in mice that are heterozygous for the orthologous factor, impeding the study of complex phenotypes and critically limiting the discovery of therapeutics. Laboratory mice have longer telomeres relative to humans, potentially protecting against age-related disease caused by haploinsufficiency. Here, we demonstrate that telomere shortening in NOTCH1-haploinsufficient mice is sufficient to elicit age-dependent cardiovascular disease involving premature calcification of the aortic valve, a phenotype that closely mimics human disease caused by NOTCH1 haploinsufficiency. Furthermore, progressive telomere shortening correlated with severity of disease, causing cardiac valve and septal disease in the neonate that was similar to the range of valve disease observed within human families. Genes that were dysregulated due to NOTCH1 haploinsufficiency in mice with shortened telomeres were concordant with proosteoblast and proinflammatory gene network alterations in human NOTCH1 heterozygous endothelial cells. These dysregulated genes were enriched for telomere-contacting promoters, suggesting a potential mechanism for telomere-dependent regulation of homeostatic gene expression. These findings reveal a critical role for telomere length in a mouse model of age-dependent human disease and provide an in vivo model in which to test therapeutic candidates targeting the progression of aortic valve disease.
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http://dx.doi.org/10.1172/JCI90338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5409071PMC
May 2017

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

Muscle Nerve 2017 09 21;56(3):522-524. Epub 2017 Mar 21.

Department of Orthopaedic Surgery, Perelman School of Medicine, The University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Introduction: The mdx 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 mdx mice.

Conclusions: This technique can benefit laboratories that require complex breeding strategies involving mdx mice. Muscle Nerve 56: 522-524, 2017.
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http://dx.doi.org/10.1002/mus.25566DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5500441PMC
September 2017

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

Cell Metab 2016 08;24(2):269-82

Department of Physiology and Institute for Diabetes, Obesity, and Metabolism, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cmet.2016.07.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4985182PMC
August 2016

Rejuvenation of the muscle stem cell population restores strength to injured aged muscles.

Nat Med 2014 Mar 16;20(3):255-64. Epub 2014 Feb 16.

Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA.

The elderly often suffer from progressive muscle weakness and regenerative failure. We demonstrate that muscle regeneration is impaired with aging owing in part to a cell-autonomous functional decline in skeletal muscle stem cells (MuSCs). Two-thirds of MuSCs from aged mice are intrinsically defective relative to MuSCs from young mice, with reduced capacity to repair myofibers and repopulate the stem cell reservoir in vivo following transplantation. This deficiency is correlated with a higher incidence of cells that express senescence markers and is due to elevated activity of the p38α and p38β mitogen-activated kinase pathway. We show that these limitations cannot be overcome by transplantation into the microenvironment of young recipient muscles. In contrast, subjecting the MuSC population from aged mice to transient inhibition of p38α and p38β in conjunction with culture on soft hydrogel substrates rapidly expands the residual functional MuSC population from aged mice, rejuvenating its potential for regeneration and serial transplantation as well as strengthening of damaged muscles of aged mice. These findings reveal a synergy between biophysical and biochemical cues that provides a paradigm for a localized autologous muscle stem cell therapy for the elderly.
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http://dx.doi.org/10.1038/nm.3464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3949152PMC
March 2014

Role of telomere dysfunction in cardiac failure in Duchenne muscular dystrophy.

Nat Cell Biol 2013 Aug 7;15(8):895-904. Epub 2013 Jul 7.

Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Institute for Stem Cell Biology and Regenerative Medicine, Clinical Sciences Research Center, Stanford University School of Medicine, Stanford, California 94305, USA.

Duchenne muscular dystrophy (DMD), the most common inherited muscular dystrophy of childhood, leads to death due to cardiorespiratory failure. Paradoxically, mdx mice with the same genetic deficiency of dystrophin exhibit minimal cardiac dysfunction, impeding the development of therapies. We postulated that the difference between mdx and DMD might result from differences in telomere lengths in mice and humans. We show here that, like DMD patients, mice that lack dystrophin and have shortened telomeres (mdx/mTR(KO)) develop severe functional cardiac deficits including ventricular dilation, contractile and conductance dysfunction, and accelerated mortality. These cardiac defects are accompanied by telomere erosion, mitochondrial fragmentation and increased oxidative stress. Treatment with antioxidants significantly retards the onset of cardiac dysfunction and death of mdx/mTR(KO) mice. In corroboration, all four of the DMD patients analysed had 45% shorter telomeres in their cardiomyocytes relative to age- and sex-matched controls. We propose that the demands of contraction in the absence of dystrophin coupled with increased oxidative stress conspire to accelerate telomere erosion culminating in cardiac failure and death. These findings provide strong support for a link between telomere length and dystrophin deficiency in the etiology of dilated cardiomyopathy in DMD and suggest preventive interventions.
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http://dx.doi.org/10.1038/ncb2790DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3774175PMC
August 2013

Short telomeres and stem cell exhaustion model Duchenne muscular dystrophy in mdx/mTR mice.

Cell 2010 Dec 9;143(7):1059-71. Epub 2010 Dec 9.

Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.

In Duchenne muscular dystrophy (DMD), dystrophin mutation leads to progressive lethal skeletal muscle degeneration. For unknown reasons, dystrophin deficiency does not recapitulate DMD in mice (mdx), which have mild skeletal muscle defects and potent regenerative capacity. We postulated that human DMD progression is a consequence of loss of functional muscle stem cells (MuSC), and the mild mouse mdx phenotype results from greater MuSC reserve fueled by longer telomeres. We report that mdx mice lacking the RNA component of telomerase (mdx/mTR) have shortened telomeres in muscle cells and severe muscular dystrophy that progressively worsens with age. Muscle wasting severity parallels a decline in MuSC regenerative capacity and is ameliorated histologically by transplantation of wild-type MuSC. These data show that DMD progression results, in part, from a cell-autonomous failure of MuSC to maintain the damage-repair cycle initiated by dystrophin deficiency. The essential role of MuSC function has therapeutic implications for DMD.
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http://dx.doi.org/10.1016/j.cell.2010.11.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3025608PMC
December 2010

Distinct roles for cell-autonomous Notch signaling in cardiomyocytes of the embryonic and adult heart.

Circ Res 2010 Feb 10;106(3):559-72. Epub 2009 Dec 10.

Mouse Biology Unit, European Molecular Biology Laboratory, Campus A. Buzzati-Traverso, Rome, Italy.

Rationale: The Notch signaling pathway is important for cell-cell communication that controls tissue formation and homeostasis during embryonic and adult life, but the precise cell targets of Notch signaling in the mammalian heart remain poorly defined.

Objective: To investigate the functional role of Notch signaling in the cardiomyocyte compartment of the embryonic and adult heart.

Methods And Results: Here, we report that either conditional overexpression of Notch1 intracellular domain (NICD1) or selective silencing of Notch signaling in the embryonic cardiomyocyte compartment results in developmental defects and perinatal lethality. In contrast, augmentation of endogenous Notch reactivation after myocardial infarction in the adult, either by inducing cardiomyocyte-specific Notch1 transgene expression or by intramyocardial delivery of a Notch1 pseudoligand, increases survival rate, improves cardiac functional performance, and minimizes fibrosis, promoting antiapoptotic and angiogenic mechanisms.

Conclusions: These results reveal a strict requirement for cell-autonomous modulation of Notch signaling during heart morphogenesis, and illustrate how the same signaling pathway that promotes congenital heart defects when perturbed in the embryo can be therapeutically redeployed for the treatment of adult myocardial damage.
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http://dx.doi.org/10.1161/CIRCRESAHA.109.203034DOI Listing
February 2010

A CREB-C/EBPbeta cascade induces M2 macrophage-specific gene expression and promotes muscle injury repair.

Proc Natl Acad Sci U S A 2009 Oct 24;106(41):17475-80. Epub 2009 Sep 24.

Mouse Biology Unit, European Molecular Biology Laboratory, Via Ramarini 32, 00015 Monterotondo, Italy.

Macrophages play an essential role in the resolution of tissue damage through removal of necrotic cells, thus paving the way for tissue regeneration. Macrophages also directly support the formation of new tissue to replace the injury, through their acquisition of an anti-inflammatory, or M2, phenotype, characterized by a gene expression program that includes IL-10, the IL-13 receptor, and arginase 1. We report that deletion of two CREB-binding sites from the Cebpb promoter abrogates Cebpb induction upon macrophage activation. This blocks the downstream induction of M2-specific Msr1, Il10, II13ra, and Arg-1 genes, whereas the inflammatory (M1) genes Il1, Il6, Tnfa, and Il12 are not affected. Mice carrying the mutated Cebpb promoter (betaDeltaCre) remove necrotic tissue from injured muscle, but exhibit severe defects in muscle fiber regeneration. Conditional deletion of the Cebpb gene in muscle cells does not affect regeneration, showing that the C/EBPbeta cascade leading to muscle repair is muscle-extrinsic. While betaDeltaCre macrophages efficiently infiltrate injured muscle they fail to upregulate Cebpb, leading to decreased Arg-1 expression. CREB-mediated induction of Cebpb expression is therefore required in infiltrating macrophages for upregulation of M2-specific genes and muscle regeneration, providing a direct genetic link between these two processes.
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http://dx.doi.org/10.1073/pnas.0908641106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2762675PMC
October 2009

Analysis of CRE-mediated recombination driven by myosin light chain 1/3 regulatory elements in embryonic and adult skeletal muscle: a tool to study fiber specification.

Genesis 2008 Aug;46(8):424-30

An increasing number of genes have been implicated in skeletal muscle fiber diversity. To study the contribution of diverse genetic elements to the regulation of fiber-type composition, we generated a transgenic mouse in which CRE recombinase expression is driven by muscle-specific regulatory sequences of the myosin light chain 1/3 locus (MLC). Using ROSA26 conditional reporter mice, we detected expression of the MLC-Cre transgene starting from embryonic day 12.5 (E12.5). By E15, recombination was detected in all muscle-derived structures. Immunohistochemical analysis revealed CRE activity was restricted to fast-twitch (type II) and excluded from slow-twitch (type I) fibers of skeletal muscle. The MLC-Cre transgenic mouse can be used in conjunction with conditional alleles to study both developmental patterning and maintenance of fast fiber-type phenotypes.
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http://dx.doi.org/10.1002/dvg.20419DOI Listing
August 2008

NF-kappaB signaling in skeletal muscle: prospects for intervention in muscle diseases.

J Mol Med (Berl) 2008 Jul 2;86(7):747-59. Epub 2008 Feb 2.

EMBL Mouse Biology Unit, Campus A. Buzzati-Traverso, 00015, Monterotondo-Scalo, Italy.

Muscle remodeling is an important physiological process that promotes adaptive changes in cytoarchitecture and protein composition after exercise, aging, or disease conditions. Numerous transcription factors have been reported to regulate skeletal muscle homeostasis. NF-kappaB is a major pleiotropic transcription factor modulating immune, inflammatory, cell survival, and proliferating responses; however, its role in muscle development, physiology, and disease has just started to be elucidated. The current review article aims to summarize the literature on the role of NF-kappaB signaling in skeletal muscle pathophysiology, investigated over the last years using in vitro and more recently in vivo systems. Understanding the exact role of NF-kappaB in muscle cells will allow better therapeutic manipulations in the setting of human muscle diseases.
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http://dx.doi.org/10.1007/s00109-008-0308-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2480606PMC
July 2008

A naturally occurring calcineurin variant inhibits FoxO activity and enhances skeletal muscle regeneration.

J Cell Biol 2007 Dec;179(6):1205-18

European Molecular Biology Laboratory (EMBL), Mouse Biology Unit, Campus Buzzatti-Traverso, Monterotondo-Scalo, 00016 Rome, Italy.

The calcium-activated phosphatase calcineurin (Cn) transduces physiological signals through intracellular pathways to influence the expression of specific genes. Here, we characterize a naturally occurring splicing variant of the CnAbeta catalytic subunit (CnAbeta1) in which the autoinhibitory domain that controls enzyme activation is replaced with a unique C-terminal region. The CnAbeta1 enzyme is constitutively active and dephosphorylates its NFAT target in a cyclosporine-resistant manner. CnAbeta1 is highly expressed in proliferating myoblasts and regenerating skeletal muscle fibers. In myoblasts, CnAbeta1 knockdown activates FoxO-regulated genes, reduces proliferation, and induces myoblast differentiation. Conversely, CnAbeta1 overexpression inhibits FoxO and prevents myotube atrophy. Supplemental CnAbeta1 transgene expression in skeletal muscle leads to enhanced regeneration, reduced scar formation, and accelerated resolution of inflammation. This unique mode of action distinguishes the CnAbeta1 isoform as a candidate for interventional strategies in muscle wasting treatment.
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http://dx.doi.org/10.1083/jcb.200704179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2140042PMC
December 2007

Targeted ablation of IKK2 improves skeletal muscle strength, maintains mass, and promotes regeneration.

J Clin Invest 2006 Nov;116(11):2945-54

European Molecular Biology Laboratory (EMBL) Mouse Biology Unit, Campus "A. Buzzati-Traverso," Monterotondo-Scalo, Italy.

NF-kappaB is a major pleiotropic transcription factor modulating immune, inflammatory, cell survival, and proliferative responses, yet the relevance of NF-kappaB signaling in muscle physiology and disease is less well documented. Here we show that muscle-restricted NF-kappaB inhibition in mice, through targeted deletion of the activating kinase inhibitor of NF-kappaB kinase 2 (IKK2), shifted muscle fiber distribution and improved muscle force. In response to denervation, IKK2 depletion protected against atrophy, maintaining fiber type, size, and strength, increasing protein synthesis, and decreasing protein degradation. IKK2-depleted mice with a muscle-specific transgene expressing a local Igf-1 isoform (mIgf-1) showed enhanced protection against muscle atrophy. In response to muscle damage, IKK2 depletion facilitated skeletal muscle regeneration through enhanced satellite cell activation and reduced fibrosis. Our results establish IKK2/NF-kappaB signaling as an important modulator of muscle homeostasis and suggest a combined role for IKK inhibitors and growth factors in the therapy of muscle diseases.
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http://dx.doi.org/10.1172/JCI28721DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1626136PMC
November 2006

IGF-1, inflammation and stem cells: interactions during muscle regeneration.

Trends Immunol 2005 Oct;26(10):535-42

EMBL Mouse Biology Unit Campus A. Buzzati-Traverso, via Ramarini 32, 00016 Monterotondo-Scalo (RM), Italy.

Insulin-like growth factor-I (IGF-1) is an important mediator in numerous developmental processes, such as proliferation, differentiation, survival, growth, apoptosis and regeneration. Mouse genetics have provided important insights into the signalling mechanisms that are necessary for the coordination of muscle repair. Recent studies on the role of IGF-1 in the promotion of cell recruitment to the injured muscle and the subsequent resolution of the inflammatory response have unveiled new perspectives into local repair mechanisms.
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http://dx.doi.org/10.1016/j.it.2005.08.002DOI Listing
October 2005

Mapping and identification of essential gene functions on the X chromosome of Drosophila.

EMBO Rep 2002 Jan 19;3(1):34-8. Epub 2001 Dec 19.

Max-Planck-Institut für Biophysikalische Chemie, Abt. Molekulare Entwicklungsbiologie, Am Fassberg, 37077 Göttingen, Germany.

The Drosophila melanogaster genome consists of four chromosomes that contain 165 Mb of DNA, 120 Mb of which are euchromatic. The two Drosophila Genome Projects, in collaboration with Celera Genomics Systems, have sequenced the genome, complementing the previously established physical and genetic maps. In addition, the Berkeley Drosophila Genome Project has undertaken large-scale functional analysis based on mutagenesis by transposable P element insertions into autosomes. Here, we present a large-scale P element insertion screen for vital gene functions and a BAC tiling map for the X chromosome. A collection of 501 X-chromosomal P element insertion lines was used to map essential genes cytogenetically and to establish short sequence tags (STSs) linking the insertion sites to the genome. The distribution of the P element integration sites, the identified genes and transcription units as well as the expression patterns of the P-element-tagged enhancers is described and discussed.
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http://dx.doi.org/10.1093/embo-reports/kvf012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1083931PMC
January 2002