Publications by authors named "Emanuele Loro"

37 Publications

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

PMO-based let-7c site blocking oligonucleotide (SBO) mediated utrophin upregulation in mdx mice, a therapeutic approach for Duchenne muscular dystrophy (DMD).

Sci Rep 2020 12 9;10(1):21492. Epub 2020 Dec 9.

Department of Physiology and Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, 755 Clinical Research Building, Philadelphia, PA, 19104, USA.

Upregulation of utrophin, a dystrophin related protein, is considered a promising therapeutic approach for Duchenne muscular dystrophy (DMD). Utrophin expression is repressed at the post-transcriptional level by a set of miRNAs, among which let-7c is evolutionarily highly conserved. We designed PMO-based SBOs complementary to the let-7c binding site in UTRN 3'UTR, with the goal of inhibiting let-7c interaction with UTRN mRNA and thus upregulating utrophin. We used the C2C12UTRN5'luc3' reporter cell line in which the 5'- and 3'-UTRs of human UTRN sequences flank luciferase, for reporter assays and the C2C12 cell line for utrophin western blots, to independently evaluate the site blocking efficiency of a series of let-7c PMOs in vitro. Treatment of one-month old mdx mice with the most effective let-7c PMO (i.e. S56) resulted in ca. two-fold higher utrophin protein expression in skeletal muscles and the improvement in dystrophic pathophysiology in mdx mice, in vivo. In summary, we show that PMO-based let-7c SBO has potential applicability for upregulating utrophin expression as a therapeutic approach for DMD.
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http://dx.doi.org/10.1038/s41598-020-76338-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7726560PMC
December 2020

Genome Editing-Mediated Utrophin Upregulation in Duchenne Muscular Dystrophy Stem Cells.

Mol Ther Nucleic Acids 2020 Dec 29;22:500-509. Epub 2020 Aug 29.

Department of Physiology and Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Utrophin upregulation is considered a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). A number of microRNAs (miRNAs) post-transcriptionally regulate utrophin expression by binding their cognate sites in the 3' UTR. Previously we have shown that miRNA: repression can be alleviated using miRNA let-7c site blocking oligonucleotides (SBOs) to achieve utrophin upregulation and functional improvement in mice. Here, we used CRISPR/Cas9-mediated genome editing to delete five miRNA binding sites (miR-150, miR-296-5p, miR-133b, let-7c, miR-196b) clustered in a 500 bp inhibitory miRNA target region (IMTR) within the 3' UTR, for achieving higher expression of endogenous utrophin. Deleting the IMTR in DMD patient-derived human induced pluripotent stem cells (DMD-hiPSCs) resulted in ca. 2-fold higher levels of utrophin protein. Differentiation of the edited DMD-hiPSCs (ΔIMTR) by MyoD overexpression resulted in increased sarcolemmal α-sarcoglycan staining consistent with improved dystrophin glycoprotein complex (DGC) restoration. These results demonstrate that CRISPR/Cas9-based genome editing offers a novel utrophin upregulation therapeutic strategy applicable to all DMD patients, irrespective of the dystrophin mutation status.
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http://dx.doi.org/10.1016/j.omtn.2020.08.031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7554652PMC
December 2020

Editorial: The Role of the Muscle Secretome in Health and Disease.

Front Physiol 2020 8;11:1101. Epub 2020 Sep 8.

Department of Promoting Health, Maternal-Infant, Excellence and Internal and Specialized Medicine "G. D'Alessandro" (PROMISE), University of Palermo, Palermo, Italy.

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http://dx.doi.org/10.3389/fphys.2020.01101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7506065PMC
September 2020

Tibetan , an allele with loss-of-function properties.

Proc Natl Acad Sci U S A 2020 06 15;117(22):12230-12238. Epub 2020 May 15.

Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104;

Tibetans have adapted to the chronic hypoxia of high altitude and display a distinctive suite of physiologic adaptations, including augmented hypoxic ventilatory response and resistance to pulmonary hypertension. Genome-wide studies have consistently identified compelling genetic signatures of natural selection in two genes of the Hypoxia Inducible Factor pathway, and The product of the former induces the degradation of the product of the latter. Key issues regarding Tibetan are whether it is a gain-of-function or loss-of-function allele, and how it might contribute to high-altitude adaptation. Tibetan PHD2 possesses two amino acid changes, D4E and C127S. We previously showed that in vitro, Tibetan PHD2 is defective in its interaction with p23, a cochaperone of the HSP90 pathway, and we proposed that Tibetan is a loss-of-function allele. Here, we report that additional PHD2 mutations at or near Asp-4 or Cys-127 impair interaction with p23 in vitro. We find that mice with the Tibetan allele display augmented hypoxic ventilatory response, supporting this loss-of-function proposal. This is phenocopied by mice with a mutation in that abrogates the PHD2:p23 interaction. haploinsufficiency, but not the Tibetan allele, ameliorates hypoxia-induced increases in right ventricular systolic pressure. The Tibetan allele is not associated with hemoglobin levels in mice. We propose that Tibetans possess genetic alterations that both activate and inhibit selective outputs of the HIF pathway to facilitate successful adaptation to the chronic hypoxia of high altitude.
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http://dx.doi.org/10.1073/pnas.1920546117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275716PMC
June 2020

Functional effects of muscle PGC-1alpha in aged animals.

Skelet Muscle 2020 05 6;10(1):14. Epub 2020 May 6.

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

PGC-1 (peroxisome-proliferator-activated receptor-γ coactivator-1) alpha is a potent transcriptional coactivator that coordinates the activation of numerous metabolic processes. Exercise strongly induces PGC-1alpha expression in muscle, and overexpression of PGC-1alpha in skeletal muscle activates mitochondrial oxidative metabolism and neovascularization, leading to markedly increased endurance. In light of these findings, PGC-1alpha has been proposed to protect from age-associated sarcopenia, bone loss, and whole-body metabolic dysfunction, although these findings have been controversial. We therefore comprehensively evaluated muscle and whole-body function and metabolism in 24-month-old transgenic mice that over-express PGC-1alpha in skeletal muscle. We find that the powerful effects of PGC-1alpha on promoting muscle oxidative capacity and protection from muscle fatigability persist in aged animals, although at the expense of muscle strength. However, skeletal muscle PGC-1alpha does not prevent bone loss and in fact accentuates it, nor does it have long-term benefit on whole-body metabolic composition or insulin sensitivity. Protection from sarcopenia is seen in male animals with overexpression of PGC-1alpha in skeletal muscle but not in female animals. In summary, muscle-specific expression of PGC-1alpha into old age has beneficial effects on muscle fatigability and may protect from sarcopenia in males, but does not improve whole-body metabolism and appears to worsen age-related trabecular bone loss.
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http://dx.doi.org/10.1186/s13395-020-00231-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7201623PMC
May 2020

Author Correction: High-throughput identification of post-transcriptional utrophin up-regulators for Duchenne muscle dystrophy (DMD) therapy.

Sci Rep 2020 Feb 28;10(1):4039. Epub 2020 Feb 28.

Department of Physiology and Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-60885-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7048851PMC
February 2020

High-throughput identification of post-transcriptional utrophin up-regulators for Duchenne muscle dystrophy (DMD) therapy.

Sci Rep 2020 02 7;10(1):2132. Epub 2020 Feb 7.

Department of Physiology and Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Upregulation of endogenous utrophin offers great promise for treating DMD, as it can functionally compensate for the lack of dystrophin caused by DMD gene mutations, without the immunogenic concerns associated with delivering dystrophin. However, post-transcriptional repression mechanisms targeting the 5' and 3' untranslated regions (UTRs) of utrophin mRNA significantly limit the magnitude of utrophin upregulation achievable by promoter activation. Using a utrophin 5'3'UTR reporter assay, we performed a high-throughput screen (HTS) for small molecules capable of relieving utrophin post-transcriptional repression. We identified 27 hits that were ranked using a using an algorithm that we designed for hit prioritization that we call Hit to Lead Prioritization Score (H2LPS). The top 10 hits were validated using an orthogonal assay for endogenous utrophin expression. Evaluation of the top scoring hit, Trichostatin A (TSA), demonstrated utrophin upregulation and functional improvement in the mdx mouse model of DMD. TSA and the other small molecules identified here represent potential starting points for DMD drug discovery efforts.
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http://dx.doi.org/10.1038/s41598-020-58737-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005813PMC
February 2020

Effect of Interleukin-15 Receptor Alpha Ablation on the Metabolic Responses to Moderate Exercise Simulated by Isometric Muscle Contractions.

Front Physiol 2019 26;10:1439. Epub 2019 Nov 26.

Department of Physiology, Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States.

Lack of interleukin 15 receptor alpha (IL15RA) increases spontaneous activity, exercise capacity and protects from diet-induced obesity by enhancing muscle energy metabolism, suggesting a role as exercise mimetic for IL15RA antagonists. Using controlled muscle stimulation mimicking moderate exercise in normal and Il15ra mice, we mapped and contrasted the metabolic pathways activated upon stimulation or deletion of IL15RA. Stimulation caused the differential regulation of 123 out of the 321 detected metabolites (FDR ≤ 0.05 and fold change ≥ ±1.5). The main energy pathways activated were fatty acid oxidation, nucleotide metabolism, and anaplerotic reactions. Notably, resting Il15ra muscles were primed in a semi-exercised state, characterized by higher pool sizes of fatty acids oxidized to support muscle activity. These studies identify the role of IL15RA in the system-wide metabolic response to exercise and should enable translational studies to harness the potential of IL15RA blockade as a novel exercise mimetic strategy.
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http://dx.doi.org/10.3389/fphys.2019.01439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6901992PMC
November 2019

New Insights into the Lactate Shuttle: Role of MCT4 in the Modulation of the Exercise Capacity.

iScience 2019 Dec 26;22:507-518. Epub 2019 Nov 26.

Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA. Electronic address:

Lactate produced by muscle during high-intensity activity is an important end product of glycolysis that supports whole body metabolism. The lactate shuttle model suggested that lactate produced by glycolytic muscle fibers is utilized by oxidative fibers. MCT4 is a proton coupled monocarboxylate transporter preferentially expressed in glycolytic muscle fibers and facilitates the lactate efflux. Here we investigated the exercise capacity of mice with disrupted lactate shuttle due to global deletion of MCT4 (MCT4) or muscle-specific deletion of the accessory protein Basigin (iMSBsg). Although MCT4 and iMSBsg mice have normal muscle morphology and contractility, only MCT4 mice exhibit an exercise intolerant phenotype. In vivo measurements of compound muscle action potentials showed a decrement in the evoked response in the MCT4 mice. This was accompanied by a significant structural degeneration of the neuromuscular junctions (NMJs). We propose that disruption of the lactate shuttle impacts motor function and destabilizes the motor unit.
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http://dx.doi.org/10.1016/j.isci.2019.11.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6920289PMC
December 2019

Non-immunogenic utrophin gene therapy for the treatment of muscular dystrophy animal models.

Nat Med 2019 10 7;25(10):1505-1511. Epub 2019 Oct 7.

Department of Surgery, Pennsylvania Muscle Institute, Penn Cardiovascular Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.

The essential product of the Duchenne muscular dystrophy (DMD) gene is dystrophin, a rod-like protein that protects striated myocytes from contraction-induced injury. Dystrophin-related protein (or utrophin) retains most of the structural and protein binding elements of dystrophin. Importantly, normal thymic expression in DMD patients should protect utrophin by central immunologic tolerance. We designed a codon-optimized, synthetic transgene encoding a miniaturized utrophin (µUtro), deliverable by adeno-associated virus (AAV) vectors. Here, we show that µUtro is a highly functional, non-immunogenic substitute for dystrophin, preventing the most deleterious histological and physiological aspects of muscular dystrophy in small and large animal models. Following systemic administration of an AAV-µUtro to neonatal dystrophin-deficient mdx mice, histological and biochemical markers of myonecrosis and regeneration are completely suppressed throughout growth to adult weight. In the dystrophin-deficient golden retriever model, µUtro non-toxically prevented myonecrosis, even in the most powerful muscles. In a stringent test of immunogenicity, focal expression of µUtro in the deletional-null German shorthaired pointer model produced no evidence of cell-mediated immunity, in contrast to the robust T cell response against similarly constructed µDystrophin (µDystro). These findings support a model in which utrophin-derived therapies might be used to treat clinical dystrophin deficiency, with a favorable immunologic profile and preserved function in the face of extreme miniaturization.
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http://dx.doi.org/10.1038/s41591-019-0594-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7274039PMC
October 2019

The HDAC3 enzymatic activity regulates skeletal muscle fuel metabolism.

J Mol Cell Biol 2019 02;11(2):133-143

Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Baylor College of Medicine, Houston, TX, USA.

Histone deacetylase 3 (HDAC3) is a major HDAC, whose enzymatic activity is targeted by small molecule inhibitors for treating a variety of conditions. However, its enzymatic activity is largely dispensable for its function in embryonic development and hepatic lipid metabolism. HDAC3 plays a pivotal role in regulating muscle fuel metabolism and contractile function. Here, we address whether these muscular functions of HDAC3 require its enzymatic activity. By mutating the NCoR/SMRT corepressors in a knock-in mouse model named NS-DADm, we ablated the enzymatic activity of HDAC3 without affecting its protein levels. Compared to the control mice, skeletal muscles from NS-DADm mice showed lower force generation, enhanced fatigue resistance, enhanced fatty acid oxidation, reduced glucose uptake during exercise, upregulated expression of metabolic genes involved in branched-chain amino acids catabolism, and reduced muscle mass during aging, without changes in the muscle fiber-type composition or mitochondrial protein content. These muscular phenotypes are similar to those observed in the HDAC3-depleted skeletal muscles, which demonstrates that, unlike that in the liver or embryonic development, the metabolic function of HDAC3 in skeletal muscles requires its enzymatic activity. These results suggest that drugs specifically targeting HDAC3 enzyme activity could be developed and tested to modulate muscle energy metabolism and exercise performance.
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http://dx.doi.org/10.1093/jmcb/mjy066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6392100PMC
February 2019

Elongated mitochondrial constrictions and fission in muscle fatigue.

J Cell Sci 2018 12 5;131(23). Epub 2018 Dec 5.

Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA 19104, USA.

Mitochondria respond to stress and undergo fusion and fission at variable rates, depending on cell status. To understand mitochondrial behavior during muscle fatigue, we investigated mitochondrial ultrastructure and expression levels of a fission- and stress-related protein in fast-twitch muscle fibers of mice subjected to fatigue testing. Mice were subjected to running at increasing speed until exhaustion at 45 min-1 h. In further experiments, high-intensity muscle stimulation through the sciatic nerve simulated the forced treadmill exercise. We detected a rare phenotype characterized by elongated mitochondrial constrictions (EMCs) connecting two separate segments of the original organelles. EMCs are rare in resting muscles and their frequency increases, albeit still at low levels, in stimulated muscles. The constrictions are accompanied by elevated phosphorylation of Drp1 (Dnm1l) at Ser 616, indicating an increased translocation of Drp1 to the mitochondrial membrane. This is indicative of a mitochondrial stress response, perhaps leading to or facilitating a long-lasting fission event. A close apposition of sarcoplasmic reticulum (SR) to the constricted areas, detected using both transmission and scanning electron microscopy, is highly suggestive of SR involvement in inducing mitochondrial constrictions.
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http://dx.doi.org/10.1242/jcs.221028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6288074PMC
December 2018

Mitochondrial ultrastructural adaptations in fast muscles of mice lacking IL15RA.

J Cell Sci 2018 11 2;131(21). Epub 2018 Nov 2.

Department of Physiology and Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA

The pro-inflammatory cytokine interleukin-15 (IL15) and its receptor α (IL15RA) participate in the regulation of musculoskeletal function and metabolism. Deletion of the gene in mice increases spontaneous activity, improves fatigue resistance in the glycolytic extensor digitorum longus (EDL) and protects from diet-induced obesity. In humans, single-nucleotide polymorphisms (SNPs) have been linked to muscle strength, metabolism and performance in elite endurance athletes. Taken together, these features suggest a possible role for IL15RA in muscle mitochondrial structure and function. Here, we have investigated the consequences of loss of IL15RA on skeletal muscle fiber-type properties and mitochondrial ultrastructure. Immunostaining of the EDL for myosin heavy chain (MyHC) isoforms revealed no significant changes in fiber type. Electron microscopy (EM) analysis of the EDL indicated an overall higher mitochondria content, and increased cristae density in subsarcolemmal and A-band mitochondrial subpopulations. The higher cristae density in mitochondria was associated with higher OPA1 and cardiolipin levels. Overall, these data extend our understanding of the role of IL15RA signaling in muscle oxidative metabolism and adaptation to exercise.
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http://dx.doi.org/10.1242/jcs.218313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6240298PMC
November 2018

Functional improvement of dystrophic muscle by repression of utrophin: let-7c interaction.

PLoS One 2017 18;12(10):e0182676. Epub 2017 Oct 18.

Department of Physiology and Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.

Duchenne muscular dystrophy (DMD) is a fatal genetic disease caused by an absence of the 427kD muscle-specific dystrophin isoform. Utrophin is the autosomal homolog of dystrophin and when overexpressed, can compensate for the absence of dystrophin and rescue the dystrophic phenotype of the mdx mouse model of DMD. Utrophin is subject to miRNA mediated repression by several miRNAs including let-7c. Inhibition of utrophin: let-7c interaction is predicted to 'repress the repression' and increase utrophin expression. We developed and tested the ability of an oligonucleotide, composed of 2'-O-methyl modified bases on a phosphorothioate backbone, to anneal to the utrophin 3'UTR and prevent let-7c miRNA binding, thereby upregulating utrophin expression and improving the dystrophic phenotype in vivo. Suppression of utrophin: let-7c interaction using bi-weekly intraperitoneal injections of let7 site blocking oligonucleotides (SBOs) for 1 month in the mdx mouse model for DMD, led to increased utrophin expression along with improved muscle histology, decreased fibrosis and increased specific force. The functional improvement of dystrophic muscle achieved using let7-SBOs suggests a novel utrophin upregulation-based therapeutic strategy for DMD.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0182676PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5646768PMC
October 2017

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue.

J Vis Exp 2017 07 6(125). Epub 2017 Jul 6.

Department of Physiology and Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania;

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include supporting mastication and deglutition, as well as taste-sensing and phonetics. Accordingly, the strength and volume of the tongue can impact the ability of vertebrates to accomplish basic activities such as feeding, communicating, and breathing. Human patients with sleep apnea have enlarged tongues, characterized by reduced muscle tone and increased intramuscular fat that can be visualized and quantified by magnetic resonance imaging (MRI). The abilities to measure force generation and viscoelastic properties of the tongue constitute important tools for obtaining functional information to correlate with imaging data. Here, we present techniques for measuring tongue force production in anesthetized Zucker rats via electrical stimulation of the hypoglossal nerves and for determining the viscoelastic properties of the tongue by applying passive Lissajous force/deformation curves.
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http://dx.doi.org/10.3791/56006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5609307PMC
July 2017

IL15RA is required for osteoblast function and bone mineralization.

Bone 2017 Oct 6;103:20-30. Epub 2017 Jun 6.

Department of Physiology and Pennsylvania Muscle Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. Electronic address:

Interleukin-15 receptor alpha (IL15RA) is an important component of interleukin-15 (IL15) pro-inflammatory signaling. In addition, IL15 and IL15RA are present in the circulation and are detected in a variety of tissues where they influence physiological functions such as muscle contractility and overall metabolism. In the skeletal system, IL15RA was previously shown to be important for osteoclastogenesis. Little is known, however, about its role in osteoblast function and bone mineralization. In this study, we evaluated bone structural and mechanical properties of an Il15ra whole-body knockout mouse (Il15ra) and used in vitro and bioinformatic analyses to understand the role IL15/IL15RA signaling on osteoblast function. We show that lack of IL15RA decreased bone mineralization in vivo and in isolated primary osteogenic cultures, suggesting a cell-autonomous effect. Il15ra osteogenic cultures also had reduced Rankl/Opg mRNA ratio, indicating defective osteoblast/osteoclast coupling. We analyzed the transcriptome of primary pre-osteoblasts from normal and Il15ra mice and identified 1150 genes that were differentially expressed at a FDR of 5%. Of these, 844 transcripts were upregulated and 306 were downregulated in Il15ra cells. The largest functional clusters, highlighted using DAVID analysis, were related to metabolism, immune response, bone mineralization and morphogenesis. The transcriptome analysis was validated by qPCR of some of the most significant hits. Using bioinformatic approaches, we identified candidate genes, including Cd200 and Enpp1, that could contribute to the reduced mineralization. Silencing Il15ra using shRNA in the calvarial osteoblast MC3T3-E1 cell line decreased ENPP1 activity. Taken together, these data support that IL15RA plays a cell-autonomous role in osteoblast function and bone mineralization.
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http://dx.doi.org/10.1016/j.bone.2017.06.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5598756PMC
October 2017

Cyclin D1 Restrains Oncogene-Induced Autophagy by Regulating the AMPK-LKB1 Signaling Axis.

Cancer Res 2017 07 18;77(13):3391-3405. Epub 2017 May 18.

Pennsylvania Cancer and Regenerative Medicine Research Center (PCARM), Doylestown, Pennsylvania.

Autophagy activated after DNA damage or other stresses mitigates cellular damage by removing damaged proteins, lipids, and organelles. Activation of the master metabolic kinase AMPK enhances autophagy. Here we report that cyclin D1 restrains autophagy by modulating the activation of AMPK. In cell models of human breast cancer or in a cyclin D1-deficient model, we observed a cyclin D1-mediated reduction in AMPK activation. Mechanistic investigations showed that cyclin D1 inhibited mitochondrial function, promoted glycolysis, and reduced activation of AMPK (pT172), possibly through a mechanism that involves cyclin D1-Cdk4/Cdk6 phosphorylation of LKB1. Our findings suggest how AMPK activation by cyclin D1 may couple cell proliferation to energy homeostasis. .
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http://dx.doi.org/10.1158/0008-5472.CAN-16-0425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5705201PMC
July 2017

Dissociation of muscle insulin sensitivity from exercise endurance in mice by HDAC3 depletion.

Nat Med 2017 Feb 19;23(2):223-234. Epub 2016 Dec 19.

Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Baylor College of Medicine, Houston, Texas, USA.

Type 2 diabetes and insulin resistance are associated with reduced glucose utilization in the muscle and poor exercise performance. Here we find that depletion of the epigenome modifier histone deacetylase 3 (HDAC3) specifically in skeletal muscle causes severe systemic insulin resistance in mice but markedly enhances endurance and resistance to muscle fatigue, despite reducing muscle force. This seemingly paradoxical phenotype is due to lower glucose utilization and greater lipid oxidation in HDAC3-depleted muscles, a fuel switch caused by the activation of anaplerotic reactions driven by AMP deaminase 3 (Ampd3) and catabolism of branched-chain amino acids. These findings highlight the pivotal role of amino acid catabolism in muscle fatigue and type 2 diabetes pathogenesis. Further, as genome occupancy of HDAC3 in skeletal muscle is controlled by the circadian clock, these results delineate an epigenomic regulatory mechanism through which the circadian clock governs skeletal muscle bioenergetics. These findings suggest that physical exercise at certain times of the day or pharmacological targeting of HDAC3 could potentially be harnessed to alter systemic fuel metabolism and exercise performance.
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http://dx.doi.org/10.1038/nm.4245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5540654PMC
February 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

Pharmacotherapy to protect the neuromuscular junction after acute organophosphorus pesticide poisoning.

Ann N Y Acad Sci 2016 06 3;1374(1):86-93. Epub 2016 Jun 3.

University of Pennsylvania, Perelman School of Medicine, Department of Physiology, Philadelphia, Pennsylvania.

Organophosphorus (OP) pesticide poisoning is a leading cause of morbidity and mortality in the developing world, affecting an estimated three million people annually. Much of the morbidity is directly related to muscle weakness, which develops 1-4 days after poisoning. This muscle weakness, termed the intermediate syndrome (IMS), leads to respiratory, bulbar, and proximal limb weakness and frequently necessitates the use of mechanical ventilation. While not entirely understood, the IMS is most likely due to persistently elevated acetylcholine (ACh), which activates nicotinic ACh receptors at the neuromuscular junction (NMJ). Thus, the NMJ is potentially a target-rich area for the development of new therapies for acute OP poisoning. In this manuscript, we discuss what is known about the IMS and studies investigating the use of nicotinic ACh receptor antagonists to prevent or mitigate NMJ dysfunction after acute OP poisoning.
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http://dx.doi.org/10.1111/nyas.13111DOI Listing
June 2016

IL-15Rα is a determinant of muscle fuel utilization, and its loss protects against obesity.

Am J Physiol Regul Integr Comp Physiol 2015 Oct 12;309(8):R835-44. Epub 2015 Aug 12.

Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania; Department of Physiology and Pennsylvania Muscle Institute, University of Pennsylvania, Philadelphia, Pennsylvania;

IL-15Rα is the widely expressed primary binding partner for IL-15. Because of the wide distribution in nonlymphoid tissues like skeletal muscle, adipose, or liver, IL-15/IL-15Rα take part in physiological and metabolic processes not directly related to immunity. In fast muscle, lack of IL-15Rα promotes an oxidative switch, with increased mitochondrial biogenesis and fatigue resistance. These effects are predicted to reproduce some of the benefits of exercise and, therefore, improve energy homeostasis. However, the direct effects of IL-15Rα on metabolism and obesity are currently unknown. We report that mice lacking IL-15Rα (IL-15Rα(-/-)) are resistant to diet-induced obesity (DIO). High-fat diet-fed IL-15Rα(-/-) mice have less body and liver fat accumulation than controls. The leaner phenotype is associated with increased energy expenditure and enhanced fatty acid oxidation by muscle mitochondria. Despite being protected against DIO, IL-15Rα(-/-) are hyperglycemic and insulin-resistant. These findings identify novel roles for IL-15Rα in metabolism and obesity.
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http://dx.doi.org/10.1152/ajpregu.00505.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4631541PMC
October 2015

Kinase-independent role of cyclin D1 in chromosomal instability and mammary tumorigenesis.

Oncotarget 2015 Apr;6(11):8525-38

Departments of Cancer Biology, Thomas Jefferson University & Hospital, Philadelphia, PA 19107, USA.

Cyclin D1 is an important molecular driver of human breast cancer but better understanding of its oncogenic mechanisms is needed, especially to enhance efforts in targeted therapeutics. Currently, pharmaceutical initiatives to inhibit cyclin D1 are focused on the catalytic component since the transforming capacity is thought to reside in the cyclin D1/CDK activity. We initiated the following study to directly test the oncogenic potential of catalytically inactive cyclin D1 in an in vivo mouse model that is relevant to breast cancer. Herein, transduction of cyclin D1(-/-) mouse embryonic fibroblasts (MEFs) with the kinase dead KE mutant of cyclin D1 led to aneuploidy, abnormalities in mitotic spindle formation, autosome amplification, and chromosomal instability (CIN) by gene expression profiling. Acute transgenic expression of either cyclin D1(WT) or cyclin D1(KE) in the mammary gland was sufficient to induce a high CIN score within 7 days. Sustained expression of cyclin D1(KE) induced mammary adenocarcinoma with similar kinetics to that of the wild-type cyclin D1. ChIP-Seq studies demonstrated recruitment of cyclin D1(WT) and cyclin D1(KE) to the genes governing CIN. We conclude that the CDK-activating function of cyclin D1 is not necessary to induce either chromosomal instability or mammary tumorigenesis.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4496164PMC
http://dx.doi.org/10.18632/oncotarget.3267DOI Listing
April 2015

Exercise protocol induces muscle, tendon, and bone adaptations in the rat shoulder.

Muscles Ligaments Tendons J 2014 Oct-Dec;4(4):413-9. Epub 2015 Feb 5.

McKay Orthopaedic Research Lab, University of Pennsylvania, Philadelphia, PA, USA.

Background: a rat model of supraspinatus overuse has suggested mechanisms governing tendon degeneration; however, delineating which changes are pathologic or simply physiologic adaptations to increased loading remains a question. The objective of this study was to develop and characterize a rat exercise model that induces systemic and local shoulder adaptations without mechanical injury to the supraspinatus tendon.

Methods: exercise rats completed a treadmill training protocol for 12 weeks. Body, fat pad, and heart weights were determined. Supraspinatus tendon collagen content, cross-sectional area, and mechanical properties were measured. Supraspinatus muscle cross-sectional area, weight, and the expression of mitochondrial oxidative phosphorylation (OXPHOS) proteins were measured. Humeri were analyzed with μCT and mechanically tested.

Results: exercise decreased fat pad mass. Supraspinatus muscle hypertrophied and had increased OXPHOS proteins. Humerus trabecular bone had increased anisotropic orientation, and cortical bone showed increased bone and tissue mineral density. Importantly, the supraspinatus tendon did not have diminished mechanical properties, indicating that this protocol was not injurious to the tendon.

Conclusion: this study establishes the first rat exercise protocol that induces adaptations in the shoulder. Future research can use this as a comparison model to study how the supraspinatus tendon adapts to loading and undergoes degeneration with overuse.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4327349PMC
March 2015

In vitro development of engineered muscle using a scaffold based on the pressure-activated microsyringe (PAM) technique.

J Tissue Eng Regen Med 2017 01 9;11(1):138-152. Epub 2014 May 9.

Research Centre 'E. Piaggio', University of Pisa, Italy.

The development of new human skeletal muscle tissue is an alternative approach to the replacement of tissue after severe damage, for example in the case of traumatic injury, where surgical reconstruction is often needed following major loss of natural tissue. Treatment to date has involved the transfer of muscle tissue from other sites, resulting in a functional loss and volume deficiency of donor sites. Approaches that seek to eliminate these problems include the relatively new solution of skeletal muscle engineering. Here there are two main components to consider: (a) the cells with their regenerative potential; and (b) the polymeric structure onto which cells are seeded and where they must perform their activities. In this paper we describe well-defined two- and three-dimensional polymeric structures able to drive the myoblast process of adhesion, proliferation and differentiation. We examine a series of polymers and protein adhesions with which to functionalize the structures, and cell-seeding methods, with a view to defining the optimal protocol for engineering skeletal muscle tissue. All polymer samples were tested for their mechanical and biological properties, to support the validity of our results in the real context of muscle tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/term.1894DOI Listing
January 2017

Cyclin D1 induction of Dicer governs microRNA processing and expression in breast cancer.

Nat Commun 2013 ;4:2812

1] Department of Cancer Biology, Thomas Jefferson University, 233 South 10th Street, Philadelphia, Pennsylvania 19107, USA [2] Kimmel Cancer Center, Thomas Jefferson University, 233 South 10th Street, Philadelphia, Pennsylvania 19107, USA [3] Research Center for Translational Medicine, Key Laboratory for Basic Research in Cardiology, East Hospital, Tongji University School of Medicine, 150 Jimo Road, Shanghai 200120, China.

Cyclin D1 encodes the regulatory subunit of a holoenzyme that phosphorylates the pRB protein and promotes G1/S cell-cycle progression and oncogenesis. Dicer is a central regulator of miRNA maturation, encoding an enzyme that cleaves double-stranded RNA or stem-loop-stem RNA into 20-25 nucleotide long small RNA, governing sequence-specific gene silencing and heterochromatin methylation. The mechanism by which the cell cycle directly controls the non-coding genome is poorly understood. Here we show that cyclin D1(-/-) cells are defective in pre-miRNA processing which is restored by cyclin D1a rescue. Cyclin D1 induces Dicer expression in vitro and in vivo. Dicer is transcriptionally targeted by cyclin D1, via a cdk-independent mechanism. Cyclin D1 and Dicer expression significantly correlates in luminal A and basal-like subtypes of human breast cancer. Cyclin D1 and Dicer maintain heterochromatic histone modification (Tri-m-H3K9). Cyclin D1-mediated cellular proliferation and migration is Dicer-dependent. We conclude that cyclin D1 induction of Dicer coordinates microRNA biogenesis.
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http://dx.doi.org/10.1038/ncomms3812DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3874416PMC
July 2014

The nuclear receptor Rev-erbα controls circadian thermogenic plasticity.

Nature 2013 Nov 27;503(7476):410-413. Epub 2013 Oct 27.

Division of Endocrinology, Diabetes, and Metabolism, Department of Medicine, Department of Genetics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA.

Circadian oscillation of body temperature is a basic, evolutionarily conserved feature of mammalian biology. In addition, homeostatic pathways allow organisms to protect their core temperatures in response to cold exposure. However, the mechanism responsible for coordinating daily body temperature rhythm and adaptability to environmental challenges is unknown. Here we show that the nuclear receptor Rev-erbα (also known as Nr1d1), a powerful transcriptional repressor, links circadian and thermogenic networks through the regulation of brown adipose tissue (BAT) function. Mice exposed to cold fare considerably better at 05:00 (Zeitgeber time 22) when Rev-erbα is barely expressed than at 17:00 (Zeitgeber time 10) when Rev-erbα is abundant. Deletion of Rev-erbα markedly improves cold tolerance at 17:00, indicating that overcoming Rev-erbα-dependent repression is a fundamental feature of the thermogenic response to cold. Physiological induction of uncoupling protein 1 (Ucp1) by cold temperatures is preceded by rapid downregulation of Rev-erbα in BAT. Rev-erbα represses Ucp1 in a brown-adipose-cell-autonomous manner and BAT Ucp1 levels are high in Rev-erbα-null mice, even at thermoneutrality. Genetic loss of Rev-erbα also abolishes normal rhythms of body temperature and BAT activity. Thus, Rev-erbα acts as a thermogenic focal point required for establishing and maintaining body temperature rhythm in a manner that is adaptable to environmental demands.
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http://dx.doi.org/10.1038/nature12642DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3839416PMC
November 2013

Cyclins and cell cycle control in cancer and disease.

Genes Cancer 2012 Nov;3(11-12):649-57

Thomas Jefferson University, Philadelphia, PA, USA.

Cyclin D1 overexpression is found in more than 50% of human breast cancers and causes mammary cancer in transgenic mice. Dysregulation of cyclin D1 gene expression or function contributes to the loss of normal cell cycle control during tumorigenesis. Recent studies have demonstrated that cyclin D1 conducts additional specific functions to regulate gene expression in the context of local chromatin, promote cellular migration, and promote chromosomal instability. It is anticipated that these additional functions contribute to the pathology associated with dysregulated cyclin D1 abundance. This article discusses evidence that examines the functional roles that cyclin D1 may play in cancer with an emphasis on other cyclin family members that also may contribute to cancer and disease in a similar fashion.
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http://dx.doi.org/10.1177/1947601913479022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3636749PMC
November 2012

Altered Ca2+ homeostasis and endoplasmic reticulum stress in myotonic dystrophy type 1 muscle cells.

Genes (Basel) 2013 Jun 4;4(2):275-92. Epub 2013 Jun 4.

Department of Neurosciences SNPSRR, University of Padova, Padova 35100, Italy.

The pathogenesis of Myotonic Dystrophy type 1 (DM1) is linked to unstable CTG repeats in the DMPK gene which induce the mis-splicing to fetal/neonatal isoforms of many transcripts, including those involved in cellular Ca2+ homeostasis. Here we monitored the splicing of three genes encoding for Ca2+ transporters and channels (RyR1, SERCA1 and CACN1S) during maturation of primary DM1 muscle cells in parallel with the functionality of the Excitation-Contraction (EC) coupling machinery. At 15 days of differentiation, fetal isoforms of SERCA1 and CACN1S mRNA were significantly higher in DM1 myotubes compared to controls. Parallel functional studies showed that the cytosolic Ca2+ response to depolarization in DM1 myotubes did not increase during the progression of differentiation, in contrast to control myotubes. While we observed no differences in the size of intracellular Ca2+ stores, DM1 myotubes showed significantly reduced RyR1 protein levels, uncoupling between the segregated ER/SR Ca2+ store and the voltage-induced Ca2+ release machinery, parallel with induction of endoplasmic reticulum (ER) stress markers. In conclusion, our data suggest that perturbed Ca2+ homeostasis, via activation of ER stress, contributes to muscle degeneration in DM1 muscle cells likely representing a premature senescence phenotype.
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http://dx.doi.org/10.3390/genes4020275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3899969PMC
June 2013

ChIP sequencing of cyclin D1 reveals a transcriptional role in chromosomal instability in mice.

J Clin Invest 2012 Mar 6;122(3):833-43. Epub 2012 Feb 6.

Department of Cancer Biology, Thomas Jefferson University and Hospital, Kimmel Cancer Center, Philadelphia, Pennsylvania 19107, USA.

Chromosomal instability (CIN) in tumors is characterized by chromosomal abnormalities and an altered gene expression signature; however, the mechanism of CIN is poorly understood. CCND1 (which encodes cyclin D1) is overexpressed in human malignancies and has been shown to play a direct role in transcriptional regulation. Here, we used genome-wide ChIP sequencing and found that the DNA-bound form of cyclin D1 occupied the regulatory region of genes governing chromosomal integrity and mitochondrial biogenesis. Adding cyclin D1 back to Ccnd1(-/-) mouse embryonic fibroblasts resulted in CIN gene regulatory region occupancy by the DNA-bound form of cyclin D1 and induction of CIN gene expression. Furthermore, increased chromosomal aberrations, aneuploidy, and centrosome abnormalities were observed in the cyclin D1-rescued cells by spectral karyotyping and immunofluorescence. To assess cyclin D1 effects in vivo, we generated transgenic mice with acute and continuous mammary gland-targeted cyclin D1 expression. These transgenic mice presented with increased tumor prevalence and signature CIN gene profiles. Additionally, interrogation of gene expression from 2,254 human breast tumors revealed that cyclin D1 expression correlated with CIN in luminal B breast cancer. These data suggest that cyclin D1 contributes to CIN and tumorigenesis by directly regulating a transcriptional program that governs chromosomal stability.
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http://dx.doi.org/10.1172/JCI60256DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3287228PMC
March 2012