Publications by authors named "Stefano Ciciliot"

31 Publications

Neuromuscular junction instability and altered intracellular calcium handling as early determinants of force loss during unloading in humans.

J Physiol 2021 Apr 21. Epub 2021 Apr 21.

Department of Biomedical Sciences, University of Padova, Padova, 35131, Italy.

Key Points: Few days of unloading are sufficient to induce a decline of skeletal muscle mass and function; notably, contractile force is lost at a faster rate than mass. The reasons behind this disproportionate loss of muscle force are still poorly understood. We provide strong evidence of two mechanisms only hypothesized until now for the rapid muscle force loss in only 10 days of bed rest. Our results show that an initial neuromuscular junction instability, accompanied by alterations in the innervation status and impairment of single fibres sarcoplasmic reticulum function contribute to the loss of contractile force in front of a preserved  myofibrillar function and central activation capacity. Early onset of neuromuscular junction instability and impairment in calcium dynamics involved in E-C coupling are proposed as eligible determinants to the greater decline in muscle force than in muscle size during unloading.

Abstract: Unloading induces rapid skeletal muscle atrophy and functional decline. Importantly, force is lost at a much higher rate than muscle mass. We aimed to investigate the early determinants of the disproportionate loss of force compared to that of muscle mass in response to unloading. Ten young participants underwent 10-day bed rest (BR). At baseline (BR0) and at 10 days (BR10), quadriceps femoris (QF) volume (VOL) and isometric maximum voluntary contraction (MVC) were assessed. At BR0 and BR10 blood samples and biopsies of vastus lateralis (VL) muscle were collected. Neuromuscular junction (NMJ) stability and myofibre innervation status were assessed, together with single fibre mechanical properties and sarcoplasmic reticulum (SR) calcium handling. From BR0 to BR10, QFVOL and MVC decreased by 5.2% (p = .003) and 14.3% (p<.001), respectively. Initial and partial denervation was detected from increased neural cell adhesion molecule (NCAM) positive myofibres at BR10 compared to BR0 (+3.4%, p = .016). NMJ instability was further inferred from increased C-terminal agrin fragment (CAF) concentration in serum (+19.2% at BR10, p = .031). Fast fibre CSA showed a trend to decrease by 15% (p = .055) at BR10, while single fibre maximal tension (force/CSA) was unchanged. However, at BR10 sarcoplasmic reticulum Ca release in response to caffeine decreased by 35.1% (P<.002) and 30.2% (P<.001) in fast and slow fibres, respectively, pointing to an impaired excitation-contraction coupling. These findings support the view that the early onset of NMJ instability and impairment in SR function are eligible mechanisms contributing to the greater decline in muscle force than in muscle size during unloading. This article is protected by copyright. All rights reserved.
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http://dx.doi.org/10.1113/JP281365DOI Listing
April 2021

Pharmacologic PPAR-γ Activation Reprograms Bone Marrow Macrophages and Partially Rescues HSPC Mobilization in Human and Murine Diabetes.

Diabetes 2020 07 28;69(7):1562-1572. Epub 2020 Apr 28.

Veneto Institute of Molecular Medicine, Padova, Italy

Mobilization of hematopoietic stem/progenitor cells (HSPC) from the bone marrow (BM) is impaired in diabetes. Excess oncostatin M (OSM) produced by M1 macrophages in the diabetic BM signals through p66Shc to induce in stromal cells and retain HSPC. BM adipocytes are another source of CXCL12 that blunts mobilization. We tested a strategy of pharmacologic macrophage reprogramming to rescue HSPC mobilization. In vitro, PPAR-γ activation with pioglitazone switched macrophages from M1 to M2, reduced expression, and prevented transcellular induction of In diabetic mice, pioglitazone treatment downregulated , , and in the hematopoietic BM, restored the effects of granulocyte-colony stimulation factor (G-CSF), and partially rescued HSPC mobilization, but it increased BM adipocytes. deletion recapitulated the effects of pioglitazone on adipogenesis, which was p66Shc independent, and double knockout of Osm and p66Shc completely rescued HSPC mobilization. In the absence of OSM, BM adipocytes produced less CXCL12, being arguably devoid of HSPC-retaining activity, whereas pioglitazone failed to downregulate in BM adipocytes. In patients with diabetes on pioglitazone therapy, HSPC mobilization after G-CSF was partially rescued. In summary, pioglitazone reprogrammed BM macrophages and suppressed OSM signaling, but sustained expression by BM adipocytes could limit full recovery of HSPC mobilization.
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http://dx.doi.org/10.2337/db19-0640DOI Listing
July 2020

Diabetes-Associated Myelopoiesis Drives Stem Cell Mobilopathy Through an OSM-p66Shc Signaling Pathway.

Diabetes 2019 06 1;68(6):1303-1314. Epub 2019 Apr 1.

Veneto Institute of Molecular Medicine (VIMM), Padova, Italy

Diabetes impairs the mobilization of hematopoietic stem/progenitor cells (HSPCs) from the bone marrow (BM), which can worsen the outcomes of HSPC transplantation and of diabetic complications. In this study, we examined the oncostatin M (OSM)-p66Shc pathway as a mechanistic link between HSPC mobilopathy and excessive myelopoiesis. We found that streptozotocin-induced diabetes in mice skewed hematopoiesis toward the myeloid lineage via hematopoietic-intrinsic p66Shc. The overexpression of resulting from myelopoiesis prevented HSPC mobilization after granulocyte colony-stimulating factor (G-CSF) stimulation. The intimate link between myelopoiesis and impaired HSPC mobilization after G-CSF stimulation was confirmed in human diabetes. Using cross-transplantation experiments, we found that deletion of in the hematopoietic or nonhematopoietic system partially rescued defective HSPC mobilization in diabetes. Additionally, mediated the diabetes-induced BM microvasculature remodeling. Ubiquitous or hematopoietic restricted deletion phenocopied deletion in preventing diabetes-associated myelopoiesis and mobilopathy. Mechanistically, we discovered that OSM couples myelopoiesis to mobilopathy by inducing in BM stromal cells via nonmitochondrial p66Shc. Altogether, these data indicate that cell-autonomous activation of the OSM-p66Shc pathway leads to diabetes-associated myelopoiesis, whereas its transcellular hematostromal activation links myelopoiesis to mobilopathy. Targeting the OSM-p66Shc pathway is a novel strategy to disconnect mobilopathy from myelopoiesis and restore normal HSPC mobilization.
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http://dx.doi.org/10.2337/db19-0080DOI Listing
June 2019

Modulation of Obesity and Insulin Resistance by the Redox Enzyme and Adaptor Protein p66.

Int J Mol Sci 2019 Feb 24;20(4). Epub 2019 Feb 24.

Veneto Institute of Molecular Medicine, 35128 Padova, Italy.

Initially reported as a longevity-related protein, the 66 kDa isoform of the mammalian locus has been implicated in several metabolic pathways, being able to act both as an adaptor protein and as a redox enzyme capable of generating reactive oxygen species (ROS) when it localizes to the mitochondrion. Ablation of p66 has been shown to be protective against obesity and the insurgence of insulin resistance, but not all the studies available in the literature agree on these points. This review will focus in particular on the role of p66 in the modulation of glucose homeostasis, obesity, body temperature, and respiration/energy expenditure. In view of the obesity and diabetes epidemic, p66 may represent a promising therapeutic target with enormous implications for human health.
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http://dx.doi.org/10.3390/ijms20040985DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6412263PMC
February 2019

Loss of mitochondrial calcium uniporter rewires skeletal muscle metabolism and substrate preference.

Cell Death Differ 2019 01 19;26(2):362-381. Epub 2018 Sep 19.

Department of Biomedical Sciences, University of Padova, 35131, Padova, Italy.

Skeletal muscle mitochondria readily accumulate Ca in response to SR store-releasing stimuli thanks to the activity of the mitochondrial calcium uniporter (MCU), the highly selective channel responsible for mitochondrial Ca uptake. MCU positively regulates myofiber size in physiological conditions and counteracts pathological loss of muscle mass. Here we show that skeletal muscle-specific MCU deletion inhibits myofiber mitochondrial Ca uptake, impairs muscle force and exercise performance, and determines a slow to fast switch in MHC expression. Mitochondrial Ca uptake is required for effective glucose oxidation, as demonstrated by the fact that in muscle-specific MCU myofibers oxidative metabolism is impaired and glycolysis rate is increased. Although defective, mitochondrial activity is partially sustained by increased fatty acid (FA) oxidation. In MCU myofibers, PDP2 overexpression drastically reduces FA dependency, demonstrating that decreased PDH activity is the main trigger of the metabolic rewiring of MCU muscles. Accordingly, PDK4 overexpression in MCU myofibers is sufficient to increase FA-dependent respiration. Finally, as a result of the muscle-specific MCU deletion, a systemic catabolic response impinging on both liver and adipose tissue metabolism occurs.
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http://dx.doi.org/10.1038/s41418-018-0191-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6329801PMC
January 2019

Transcriptional programming of lipid and amino acid metabolism by the skeletal muscle circadian clock.

PLoS Biol 2018 08 10;16(8):e2005886. Epub 2018 Aug 10.

Helmholtz Diabetes Center (HMGU) and German Center for Diabetes Research (DZD), Institute for Diabetes and Obesity (IDO), Munich, Germany.

Circadian clocks are fundamental physiological regulators of energy homeostasis, but direct transcriptional targets of the muscle clock machinery are unknown. To understand how the muscle clock directs rhythmic metabolism, we determined genome-wide binding of the master clock regulators brain and muscle ARNT-like protein 1 (BMAL1) and REV-ERBα in murine muscles. Integrating occupancy with 24-hr gene expression and metabolomics after muscle-specific loss of BMAL1 and REV-ERBα, here we unravel novel molecular mechanisms connecting muscle clock function to daily cycles of lipid and protein metabolism. Validating BMAL1 and REV-ERBα targets using luciferase assays and in vivo rescue, we demonstrate how a major role of the muscle clock is to promote diurnal cycles of neutral lipid storage while coordinately inhibiting lipid and protein catabolism prior to awakening. This occurs by BMAL1-dependent activation of Dgat2 and REV-ERBα-dependent repression of major targets involved in lipid metabolism and protein turnover (MuRF-1, Atrogin-1). Accordingly, muscle-specific loss of BMAL1 is associated with metabolic inefficiency, impaired muscle triglyceride biosynthesis, and accumulation of bioactive lipids and amino acids. Taken together, our data provide a comprehensive overview of how genomic binding of BMAL1 and REV-ERBα is related to temporal changes in gene expression and metabolite fluctuations.
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http://dx.doi.org/10.1371/journal.pbio.2005886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6105032PMC
August 2018

Interplay between gut microbiota and p66Shc affects obesity-associated insulin resistance.

FASEB J 2018 07 21;32(7):4004-4015. Epub 2018 Feb 21.

Venetian Institute of Molecular Medicine, Padua, Italy.

The 66 kDa isoform of the mammalian Shc gene promotes adipogenesis, and p66Shc mice accumulate less body weight than wild-type (WT) mice. As the metabolic consequences of the leaner phenotype of p66Shc mice is debated, we hypothesized that gut microbiota may be involved. We confirmed that p66Shc mice gained less weight than WT mice when on a high-fat diet (HFD), but they were not protected from insulin resistance and glucose intolerance. p66Shc deletion significantly modified the composition of gut microbiota and their modification after an HFD. This was associated with changes in gene expression of Il-1b and regenerating islet-derived protein 3 γ ( Reg3g) in the gut and in systemic trimethylamine N-oxide and branched chain amino acid levels, despite there being no difference in intestinal structure and permeability. Depleting gut microbiota at the end of HFD rendered both strains more glucose tolerant but improved insulin sensitivity only in p66Shc mice. Microbiota-depleted WT mice cohoused with microbiota-competent p66Shc mice became significantly more insulin resistant than WT mice cohoused with WT mice, despite no difference in weight gain. These findings reconcile previous inconsistent observations on the metabolic phenotype of p66Shc mice and illustrate the complex microbiome-host-genotype interplay under metabolic stress.-Ciciliot, S., Albiero, M., Campanaro, S., Poncina, N., Tedesco, S., Scattolini, V., Dalla Costa, F., Cignarella, A., Vettore, M., Di Gangi, I. M., Bogialli, S., Avogaro, A., Fadini, G. P. Interplay between gut microbiota and p66Shc affects obesity-associated insulin resistance.
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http://dx.doi.org/10.1096/fj.201701409RDOI Listing
July 2018

Single Muscle Fiber Proteomics Reveals Fiber-Type-Specific Features of Human Muscle Aging.

Cell Rep 2017 06;19(11):2396-2409

Max-Planck-Institute of Biochemistry, Martinsried 82152, Germany. Electronic address:

Skeletal muscle is a key tissue in human aging, which affects different muscle fiber types unequally. We developed a highly sensitive single muscle fiber proteomics workflow to study human aging and show that the senescence of slow and fast muscle fibers is characterized by diverging metabolic and protein quality control adaptations. Whereas mitochondrial content declines with aging in both fiber types, glycolysis and glycogen metabolism are upregulated in slow but downregulated in fast muscle fibers. Aging mitochondria decrease expression of the redox enzyme monoamine oxidase A. Slow fibers upregulate a subset of actin and myosin chaperones, whereas an opposite change happens in fast fibers. These changes in metabolism and sarcomere quality control may be related to the ability of slow, but not fast, muscle fibers to maintain their mass during aging. We conclude that single muscle fiber analysis by proteomics can elucidate pathophysiology in a sub-type-specific manner.
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http://dx.doi.org/10.1016/j.celrep.2017.05.054DOI Listing
June 2017

Age-Associated Loss of OPA1 in Muscle Impacts Muscle Mass, Metabolic Homeostasis, Systemic Inflammation, and Epithelial Senescence.

Cell Metab 2017 Jun 25;25(6):1374-1389.e6. Epub 2017 May 25.

Venetian Institute of Molecular Medicine, via Orus 2, 35129 Padova, Italy; Department of Biomedical Science, University of Padova, via G. Colombo 3, 35100 Padova, Italy; Department of Medicine, McGill University, Montreal, QC H4A 3J1, Canada. Electronic address:

Mitochondrial dysfunction occurs during aging, but its impact on tissue senescence is unknown. Here, we find that sedentary but not active humans display an age-related decline in the mitochondrial protein, optic atrophy 1 (OPA1), that is associated with muscle loss. In adult mice, acute, muscle-specific deletion of Opa1 induces a precocious senescence phenotype and premature death. Conditional and inducible Opa1 deletion alters mitochondrial morphology and function but not DNA content. Mechanistically, the ablation of Opa1 leads to ER stress, which signals via the unfolded protein response (UPR) and FoxOs, inducing a catabolic program of muscle loss and systemic aging. Pharmacological inhibition of ER stress or muscle-specific deletion of FGF21 compensates for the loss of Opa1, restoring a normal metabolic state and preventing muscle atrophy and premature death. Thus, mitochondrial dysfunction in the muscle can trigger a cascade of signaling initiated at the ER that systemically affects general metabolism and aging.
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http://dx.doi.org/10.1016/j.cmet.2017.04.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5462533PMC
June 2017

Microgravity-Induced Transcriptome Adaptation in Mouse Paraspinal Muscle Highlights Insulin Resistance-Linked Genes.

Front Physiol 2017 5;8:279. Epub 2017 May 5.

Center of Space Medicine Berlin, Charité Universitätsmedizin BerlinBerlin, Germany.

Microgravity as well as chronic muscle disuse are two causes of low back pain originated at least in part from paraspinal muscle deconditioning. At present no study investigated the complexity of the molecular changes in human or mouse paraspinal muscles exposed to microgravity. The aim of this study was to evaluate adaptation to microgravity at both morphological and global gene expression level. C57BL/N6 male mice were flown aboard the BION-M1 biosatellite for 30 days (BF) or housed in a replicate flight habitat on ground (BG). Myofiber cross sectional area and myosin heavy chain subtype patterns were respectively not or slightly altered in of BF mice. Global gene expression analysis identified 89 transcripts differentially regulated in of BF vs. BG mice. Microgravity-induced gene expression changes of lipocalin 2 (Lcn2), sestrin 1(Sesn1), phosphatidylinositol 3-kinase, regulatory subunit polypeptide 1 (p85 alpha) (Pik3r1), v-maf musculoaponeurotic fibrosarcoma oncogene family protein B (Mafb), protein kinase C delta (Prkcd), Muscle Atrophy F-box (MAFbx/Atrogin-1/Fbxo32), and Muscle RING Finger 1 (MuRF-1) were further validated by real time qPCR analysis. In conclusion, our study highlighted the regulation of transcripts mainly linked to insulin sensitivity and metabolism in following 30 days of microgravity exposure. The apparent absence of robust signs of back muscle atrophy in space-flown mice, despite the overexpression of Atrogin-1 and MuRF-1, opens new questions on the possible role of microgravity-sensitive genes in the regulation of peripheral insulin resistance following unloading and its consequences on paraspinal skeletal muscle physiology.
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http://dx.doi.org/10.3389/fphys.2017.00279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5418220PMC
May 2017

Gene Expression Profiling in Slow-Type Calf Soleus Muscle of 30 Days Space-Flown Mice.

PLoS One 2017 11;12(1):e0169314. Epub 2017 Jan 11.

Center for Space Medicine Berlin, Neuromuscular Group, Charité Universitätsmedizin Berlin, Berlin, Germany.

Microgravity exposure as well as chronic disuse are two main causes of skeletal muscle atrophy in animals and humans. The antigravity calf soleus is a reference postural muscle to investigate the mechanism of disuse-induced maladaptation and plasticity of human and rodent (rats or mice) skeletal musculature. Here, we report microgravity-induced global gene expression changes in space-flown mouse skeletal muscle and the identification of yet unknown disuse susceptible transcripts found in soleus (a mainly slow phenotype) but not in extensor digitorum longus (a mainly fast phenotype dorsiflexor as functional counterpart to soleus). Adult C57Bl/N6 male mice (n = 5) flew aboard a biosatellite for 30 days on orbit (BION-M1 mission, 2013), a sex and age-matched cohort were housed in standard vivarium cages (n = 5), or in a replicate flight habitat as ground control (n = 5). Next to disuse atrophy signs (reduced size and myofiber phenotype I to II type shift) as much as 680 differentially expressed genes were found in the space-flown soleus, and only 72 in extensor digitorum longus (only 24 genes in common) compared to ground controls. Altered expression of gene transcripts matched key biological processes (contractile machinery, calcium homeostasis, muscle development, cell metabolism, inflammatory and oxidative stress response). Some transcripts (Fzd9, Casq2, Kcnma1, Ppara, Myf6) were further validated by quantitative real-time PCR (qRT-PCR). Besides previous reports on other leg muscle types we put forth for the first time a complete set of microgravity susceptible gene transcripts in soleus of mice as promising new biomarkers or targets for optimization of physical countermeasures and rehabilitation protocols to overcome disuse atrophy conditions in different clinical settings, rehabilitation and spaceflight.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0169314PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5226721PMC
August 2017

MRF4 negatively regulates adult skeletal muscle growth by repressing MEF2 activity.

Nat Commun 2016 08 3;7:12397. Epub 2016 Aug 3.

Venetian Institute of Molecular Medicine (VIMM), via Orus 2, 35129 Padova, Italy.

The myogenic regulatory factor MRF4 is highly expressed in adult skeletal muscle but its function is unknown. Here we show that Mrf4 knockdown in adult muscle induces hypertrophy and prevents denervation-induced atrophy. This effect is accompanied by increased protein synthesis and widespread activation of muscle-specific genes, many of which are targets of MEF2 transcription factors. MEF2-dependent genes represent the top-ranking gene set enriched after Mrf4 RNAi and a MEF2 reporter is inhibited by co-transfected MRF4 and activated by Mrf4 RNAi. The Mrf4 RNAi-dependent increase in fibre size is prevented by dominant negative MEF2, while constitutively active MEF2 is able to induce myofibre hypertrophy. The nuclear localization of the MEF2 corepressor HDAC4 is impaired by Mrf4 knockdown, suggesting that MRF4 acts by stabilizing a repressor complex that controls MEF2 activity. These findings open new perspectives in the search for therapeutic targets to prevent muscle wasting, in particular sarcopenia and cachexia.
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http://dx.doi.org/10.1038/ncomms12397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4976255PMC
August 2016

Regulatory T cells and skeletal muscle regeneration.

FEBS J 2017 02 18;284(4):517-524. Epub 2016 Aug 18.

Division of Immunology, Transplantation and Infectious Disease, San Raffaele Scientific Institute, Vita-Salute San Raffaele University, Milano, Italy.

Skeletal muscle regeneration results from the activation and differentiation of myogenic stem cells, called satellite cells, located beneath the basal lamina of the muscle fibers. Inflammatory and immune cells have a crucial role in the regeneration process. Acute muscle injury causes an immediate transient wave of neutrophils followed by a more persistent infiltration of M1 (proinflammatory) and M2 (anti-inflammatory/proregenerative) macrophages. New studies show that injured muscle is also infiltrated by a specialized population of regulatory T (Treg) cells, which control both the inflammatory response, by promoting the M1-to-M2 switch, and the activation of satellite cells. Treg cells accumulate in injured muscle in response to specific cytokines, such as IL-33, and promote muscle growth by releasing growth factors, such as amphiregulin. Muscle repair during aging is impaired due to reduced number of Treg cells and can be enhanced by IL-33 supplementation. Migration of Treg cells could also contribute to explain the effect of heterochronic parabiosis, whereby muscle regeneration of aged mice can be improved by a parabiotically linked young partners. In mdx dystrophin-deficient mice, a model of human Duchenne muscular dystrophy, muscle injury, and inflammation is mitigated by expansion of the Treg-cell population but exacerbated by Treg-cell depletion. These findings support the notion that immunological mechanisms are not only essential in the response to pathogenic microbes and tumor cells but also have a wider homeostatic role in tissue repair, and open new perspectives for boosting muscle growth in chronic muscle disease and during aging.
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http://dx.doi.org/10.1111/febs.13827DOI Listing
February 2017

Concise Review: Perspectives and Clinical Implications of Bone Marrow and Circulating Stem Cell Defects in Diabetes.

Stem Cells 2017 01 11;35(1):106-116. Epub 2016 Jul 11.

Department of Medicine, University of Padova, and Venetian Institute of Molecular Medicine, Padova, 35128, Italy.

Diabetes mellitus is a complex systemic disease characterized by severe morbidity and excess mortality. The burden of its multiorgan complications relies on an imbalance between hyperglycemic cell damage and defective endogenous reparative mechanisms. Inflammation and abnormalities in several hematopoietic components are typically found in diabetes. The discovery that diabetes reduces circulating stem/progenitor cells and impairs their function has opened an entire new field of study where diabetology comes into contact with hematology and regenerative medicine. It is being progressively recognized that such rare circulating cell populations mirror finely regulated processes involved in hematopoiesis, immunosurveillance, and peripheral tissue homeostasis. From a clinical perspective, pauperization of circulating stem cells predicts adverse outcomes and death. Furthermore, studies in murine models and humans have identified the bone marrow (BM) as a previously neglected site of diabetic end-organ damage, characterized by microangiopathy, neuropathy, fat deposition, and inflammation. As a result, diabetes impairs the mobilization of BM stem/progenitor cells, a defect known as mobilopathy or myelokathexis, with negative consequences for physiologic hematopoiesis, immune regulation, and tissue regeneration. A better understanding of the molecular and cellular processes that govern the BM stem cell niche, cell mobilization, and kinetics in peripheral tissues may uncover new therapeutic strategies for patients with diabetes. This concise review summarizes the current knowledge on the interplay between the BM, circulating stem cells, and diabetes, and sets the stages for future developments in the field. Stem Cells 2017;35:106-116.
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http://dx.doi.org/10.1002/stem.2445DOI Listing
January 2017

NETosis Delays Diabetic Wound Healing in Mice and Humans.

Diabetes 2016 04 6;65(4):1061-71. Epub 2016 Jan 6.

Department of Medicine, University of Padova, Padova, Italy Venetian Institute of Molecular Medicine, Padova, Italy.

Upon activation, neutrophils undergo histone citrullination by protein arginine deiminase (PAD)4, exocytosis of chromatin and enzymes as neutrophil extracellular traps (NETs), and death. In diabetes, neutrophils are primed to release NETs and die by NETosis. Although this process is a defense against infection, NETosis can damage tissue. Therefore, we examined the effect of NETosis on the healing of diabetic foot ulcers (DFUs). Using proteomics, we found that NET components were enriched in nonhealing human DFUs. In an independent validation cohort, a high concentration of neutrophil elastase in the wound was associated with infection and a subsequent worsening of the ulcer. NET components (elastase, histones, neutrophil gelatinase-associated lipocalin, and proteinase-3) were elevated in the blood of patients with DFUs. Circulating elastase and proteinase-3 were associated with infection, and serum elastase predicted delayed healing. Neutrophils isolated from the blood of DFU patients showed an increased spontaneous NETosis but an impaired inducible NETosis. In mice, skin PAD4 activity was increased by diabetes, and FACS detection of histone citrullination, together with intravital microscopy, showed that NETosis occurred in the bed of excisional wounds. PAD4 inhibition by Cl-amidine reduced NETting neutrophils and rescued wound healing in diabetic mice. Cumulatively, these data suggest that NETosis delays DFU healing.
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http://dx.doi.org/10.2337/db15-0863DOI Listing
April 2016

The calcineurin-NFAT pathway controls activity-dependent circadian gene expression in slow skeletal muscle.

Mol Metab 2015 Nov 25;4(11):823-33. Epub 2015 Sep 25.

Venetian Institute of Molecular Medicine (VIMM), Padova, Italy ; Department of Biomedical Sciences, University of Padova, Italy.

Objective: Physical activity and circadian rhythms are well-established determinants of human health and disease, but the relationship between muscle activity and the circadian regulation of muscle genes is a relatively new area of research. It is unknown whether muscle activity and muscle clock rhythms are coupled together, nor whether activity rhythms can drive circadian gene expression in skeletal muscle.

Methods: We compared the circadian transcriptomes of two mouse hindlimb muscles with vastly different circadian activity patterns, the continuously active slow soleus and the sporadically active fast tibialis anterior, in the presence or absence of a functional skeletal muscle clock (skeletal muscle-specific Bmal1 KO). In addition, we compared the effect of denervation on muscle circadian gene expression.

Results: We found that different skeletal muscles exhibit major differences in their circadian transcriptomes, yet core clock gene oscillations were essentially identical in fast and slow muscles. Furthermore, denervation caused relatively minor changes in circadian expression of most core clock genes, yet major differences in expression level, phase and amplitude of many muscle circadian genes.

Conclusions: We report that activity controls the oscillation of around 15% of skeletal muscle circadian genes independently of the core muscle clock, and we have identified the Ca(2+)-dependent calcineurin-NFAT pathway as an important mediator of activity-dependent circadian gene expression, showing that circadian locomotor activity rhythms drive circadian rhythms of NFAT nuclear translocation and target gene expression.
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http://dx.doi.org/10.1016/j.molmet.2015.09.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4632177PMC
November 2015

p66Shc deletion or deficiency protects from obesity but not metabolic dysfunction in mice and humans.

Diabetologia 2015 Oct 30;58(10):2352-60. Epub 2015 Jun 30.

Department of Medicine, University of Padua, Via Giustiniani, 2, 35128, Padua, Italy.

Aims/hypothesis: Oxygen radicals generated by p66Shc drive adipogenesis, but contradictory data exist on the role of p66Shc in the development of obesity and the metabolic syndrome. We herein explored the relationships among p66Shc, adipose tissue remodelling and glucose metabolism using mouse models and human adipose tissue samples.

Methods: In wild-type (WT), leptin-deficient (ob/ob), p66Shc(-/-) and p66Shc(-/-) ob/ob mice up to 30 weeks of age, we analysed body weight, subcutaneous and visceral adipose tissue histopathology, glucose tolerance and insulin sensitivity, and liver and muscle fat accumulation. A group of mice on a high fat diet (HFD) was also analysed. A parallel study was conducted on adipose tissue collected from patients undergoing elective surgery.

Results: We found that p66Shc(-/-) mice were slightly leaner than WT mice, and p66Shc(-/-) ob/ob mice became less obese than ob/ob mice. Despite their lower body weight, p66Shc(-/-) mice accumulated ectopic fat in the liver and muscles, and were glucose intolerant and insulin resistant. Features of adverse adipose tissue remodelling induced by obesity, including adipocyte enlargement, apoptosis, inflammation and perfusion were modestly and transiently improved by p66Shc (also known as Shc1) deletion. After 12 weeks of the HFD, p66Shc(-/-) mice were leaner than but equally glucose intolerant and insulin resistant compared with WT mice. In 77 patients, we found a direct correlation between BMI and p66Shc protein levels. Patients with low p66Shc levels were less obese, but were not protected from other metabolic syndrome features (diabetes, dyslipidaemia and hypertension).

Conclusions/interpretation: In mice and humans, reduced p66Shc levels protect from obesity, but not from ectopic fat accumulation, glucose intolerance and insulin resistance.
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http://dx.doi.org/10.1007/s00125-015-3667-8DOI Listing
October 2015

Bone Marrow Macrophages Contribute to Diabetic Stem Cell Mobilopathy by Producing Oncostatin M.

Diabetes 2015 Aug 24;64(8):2957-68. Epub 2015 Mar 24.

Department of Medicine, University of Padova, Padova, Italy Venetian Institute of Molecular Medicine, Padova, Italy

Diabetes affects bone marrow (BM) structure and impairs mobilization of stem cells (SCs) into peripheral blood (PB). This amplifies multiorgan complications because BMSCs promote vascular repair. Because diabetes skews macrophage phenotypes and BM macrophages (BMMΦ) prevent SC mobilization, we hypothesized that excess BMMΦ contribute to diabetic SC mobilopathy. We show that patients with diabetes have increased M1 macrophages, whereas diabetic mice have increased CD169(+) BMMΦ with SC-retaining activity. Depletion of BMMΦ restored SC mobilization in diabetic mice. We found that CD169 labels M1 macrophages and that conditioned medium (CM) from M1 macrophages, but not from M0 and M2 macrophages, induced chemokine (C-X-C motif) ligand 12 (CXCL12) expression by mesenchymal stem/stromal cells. In silico data mining and in vitro validation identified oncostatin M (OSM) as the soluble mediator contained in M1 CM that induces CXCL12 expression via a mitogen-activated protein kinase kinase-p38-signal transducer and activator of a transcription 3-dependent pathway. In diabetic mice, OSM neutralization prevented CXCL12 induction and improved granulocyte-colony stimulating factor and ischemia-induced mobilization, SC homing to ischemic muscles, and vascular recovery. In patients with diabetes, BM plasma OSM levels were higher and correlated with the BM-to-PB SC ratio. In conclusion, BMMΦ prevent SC mobilization by OSM secretion, and OSM antagonism is a strategy to restore BM function in diabetes, which can translate into protection mediated by BMSCs.
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http://dx.doi.org/10.2337/db14-1473DOI Listing
August 2015

NETosis is induced by high glucose and associated with type 2 diabetes.

Acta Diabetol 2015 Jun 12;52(3):497-503. Epub 2014 Nov 12.

Department of Medicine, University of Padova, Via Giustiniani, 2, 35128, Padua, Italy.

Aims: The role of neutrophils in diabetes and its complications is unclear. Upon challenge with microbes and inflammatory triggers, neutrophils release enzymes and nuclear material, forming neutrophils extracellular traps (NETs) and thereby dying by NETosis. We herein tested NET formation and NETosis products in high glucose and in the setting of type 2 diabetes (T2D).

Methods: NETosis was assessed in vitro in cells exposed to 0, 5, 25 mM glucose and 25 mM mannitol, DMSO and PMA using immunofluorescence staining for elastase, DNA and chromatin. Single-cell morphometric analysis was used to detect enter of elastase in the nucleus and extrusion of nuclear material. Release of NETs was quantified by staining with Hoechst 33342. In 38 T2D and 38 age- and sex-matched non-diabetic individuals, we determined plasma elastase, mono- and oligonucleosomes and double-strand (ds) DNA, as circulating NETosis products.

Results: NETosis was accurately reproduced in vitro: high (25 mM) glucose increased NETosis rate and release of NETs compared with 5 mM glucose and 25 mM mannitol. T2D patients showed increased plasma elastase, mono- and oligonucleosomes and dsDNA compared with non-diabetic control individuals. A positive correlation was found between HbA1c and mono- and oligonucleosomes, whereas dsDNA was correlated with the presence of nephropathy and cardiovascular disease. Serum IL-6 concentrations were higher in T2D compared with CTRL and correlated with serum dsDNA levels.

Conclusions: High glucose and hyperglycemia increase release of NETs and circulating markers of NETosis, respectively. This finding provides a link among neutrophils, inflammation and tissue damage in diabetes.
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http://dx.doi.org/10.1007/s00592-014-0676-xDOI Listing
June 2015

Vascular smooth muscle cells and monocyte-macrophages accomplice in the accelerated atherosclerosis of insulin resistance states.

Cardiovasc Res 2014 Jul 5;103(2):194-5. Epub 2014 Jun 5.

Venetian Institute of Molecular Medicine, Padova, Italy Department of Medicine, University of Padova, Via Giustiniani, 2, 35100 Padova, Italy.

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http://dx.doi.org/10.1093/cvr/cvu144DOI Listing
July 2014

Muscle insulin sensitivity and glucose metabolism are controlled by the intrinsic muscle clock.

Mol Metab 2014 Feb 23;3(1):29-41. Epub 2013 Oct 23.

Venetian Institute of Molecular Medicine, Via Orus 2, 35129 Padova, Italy ; CNR Institute of Neuroscience, Padova, Italy.

Circadian rhythms control metabolism and energy homeostasis, but the role of the skeletal muscle clock has never been explored. We generated conditional and inducible mouse lines with muscle-specific ablation of the core clock gene Bmal1. Skeletal muscles from these mice showed impaired insulin-stimulated glucose uptake with reduced protein levels of GLUT4, the insulin-dependent glucose transporter, and TBC1D1, a Rab-GTPase involved in GLUT4 translocation. Pyruvate dehydrogenase (PDH) activity was also reduced due to altered expression of circadian genes Pdk4 and Pdp1, coding for PDH kinase and phosphatase, respectively. PDH inhibition leads to reduced glucose oxidation and diversion of glycolytic intermediates to alternative metabolic pathways, as revealed by metabolome analysis. The impaired glucose metabolism induced by muscle-specific Bmal1 knockout suggests that a major physiological role of the muscle clock is to prepare for the transition from the rest/fasting phase to the active/feeding phase, when glucose becomes the predominant fuel for skeletal muscle.
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http://dx.doi.org/10.1016/j.molmet.2013.10.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3929910PMC
February 2014

Diabetes causes bone marrow autonomic neuropathy and impairs stem cell mobilization via dysregulated p66Shc and Sirt1.

Diabetes 2014 Apr 22;63(4):1353-65. Epub 2013 Nov 22.

Venetian Institute of Molecular Medicine, Padova, Italy.

Diabetes compromises the bone marrow (BM) microenvironment and reduces the number of circulating CD34(+) cells. Diabetic autonomic neuropathy (DAN) may impact the BM, because the sympathetic nervous system is prominently involved in BM stem cell trafficking. We hypothesize that neuropathy of the BM affects stem cell mobilization and vascular recovery after ischemia in patients with diabetes. We report that, in patients, cardiovascular DAN was associated with fewer circulating CD34(+) cells. Experimental diabetes (streptozotocin-induced and ob/ob mice) or chemical sympathectomy in mice resulted in BM autonomic neuropathy, impaired Lin(-)cKit(+)Sca1(+) (LKS) cell and endothelial progenitor cell (EPC; CD34(+)Flk1(+)) mobilization, and vascular recovery after ischemia. DAN increased the expression of the 66-kDa protein from the src homology and collagen homology domain (p66Shc) and reduced the expression of sirtuin 1 (Sirt1) in mice and humans. p66Shc knockout (KO) in diabetic mice prevented DAN in the BM, and rescued defective LKS cell and EPC mobilization. Hematopoietic Sirt1 KO mimicked the diabetic mobilization defect, whereas hematopoietic Sirt1 overexpression in diabetes rescued defective mobilization and vascular repair. Through p66Shc and Sirt1, diabetes and sympathectomy elevated the expression of various adhesion molecules, including CD62L. CD62L KO partially rescued the defective stem/progenitor cell mobilization. In conclusion, autonomic neuropathy in the BM impairs stem cell mobilization in diabetes with dysregulation of the life-span regulators p66Shc and Sirt1.
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http://dx.doi.org/10.2337/db13-0894DOI Listing
April 2014

Effects of pleiotrophin overexpression on mouse skeletal muscles in normal loading and in actual and simulated microgravity.

PLoS One 2013 28;8(8):e72028. Epub 2013 Aug 28.

Section of Pharmacology, Department of Pharmacy & Drug Sciences, University of Bari - Aldo Moro, Bari, Italy.

Pleiotrophin (PTN) is a widespread cytokine involved in bone formation, neurite outgrowth, and angiogenesis. In skeletal muscle, PTN is upregulated during myogenesis, post-synaptic induction, and regeneration after crushing, but little is known regarding its effects on muscle function. Here, we describe the effects of PTN on the slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles in mice over-expressing PTN under the control of a bone promoter. The mice were maintained in normal loading or disuse condition, induced by hindlimb unloading (HU) for 14 days. Effects of exposition to near-zero gravity during a 3-months spaceflight (SF) into the Mice Drawer System are also reported. In normal loading, PTN overexpression had no effect on muscle fiber cross-sectional area, but shifted soleus muscle toward a slower phenotype, as shown by an increased number of oxidative type 1 fibers, and increased gene expression of cytochrome c oxidase subunit IV and citrate synthase. The cytokine increased soleus and EDL capillary-to-fiber ratio. PTN overexpression did not prevent soleus muscle atrophy, slow-to-fast transition, and capillary regression induced by SF and HU. Nevertheless, PTN exerted various effects on sarcolemma ion channel expression/function and resting cytosolic Ca(2+) concentration in soleus and EDL muscles, in normal loading and after HU. In conclusion, the results show very similar effects of HU and SF on mouse soleus muscle, including activation of specific gene programs. The EDL muscle is able to counterbalance this latter, probably by activating compensatory mechanisms. The numerous effects of PTN on muscle gene expression and functional parameters demonstrate the sensitivity of muscle fibers to the cytokine. Although little benefit was found in HU muscle disuse, PTN may emerge useful in various muscle diseases, because it exerts synergetic actions on muscle fibers and vessels, which could enforce oxidative metabolism and ameliorate muscle performance.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0072028PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3756024PMC
May 2014

Myeloid calcifying cells promote atherosclerotic calcification via paracrine activity and allograft inflammatory factor-1 overexpression.

Basic Res Cardiol 2013 Jul 26;108(4):368. Epub 2013 Jun 26.

Venetian Institute of Molecular Medicine, Padova, Italy.

Several cell types contribute to atherosclerotic calcification. Myeloid calcifying cells (MCCs) are monocytes expressing osteocalcin (OC) and bone alkaline phosphatase (BAP). Herein, we tested whether MCCs promote atherosclerotic calcification in vivo. We show that the murine spleen contains OC(+)BAP(+) cells with a phenotype similar to human MCCs, a high expression of adhesion molecules and CD11b, and capacity to calcify in vitro and in vivo. Injection of GFP(+) OC(+)BAP(+) cells into 8- or 40-week ApoE(-/-) mice led to more extensive calcifications in atherosclerotic areas after 24 or 4 weeks, respectively, compared to control OC(-)BAP(-) cells. Despite that OC(+)BAP(+) cells had a selective transendothelial migration capacity, tracking of the GFP signal revealed that presence of injected cells within atherosclerotic areas was an extremely rare event and so GFP mRNA was undetectable by qPCR of lesion extracts. By converse, injected OC(+)BAP(+) cells persisted in the bloodstream and bone marrow up to 24 weeks, suggesting a paracrine effect. Indeed, OC(+)BAP(+) cell-conditioned medium (CM) promoted calcification by cultured vascular smooth muscle cells (VSMC) more than CM from OC(-)BAP(-) cells. A genomic and proteomic investigation of MCCs identified allograft inflammatory factor (AIF)-1 as a potential candidate of this paracrine activity. AIF-1 stimulated VSMC calcification in vitro and monocyte-specific (CD11b-driven) AIF-1 overexpression in ApoE(-/-) mice increased calcium content in atherosclerotic areas. In conclusion, we show that murine OC(+)BAP(+) cells correspond to human MCCs and promote atherosclerotic calcification in ApoE(-/-) mice, through paracrine activity and modulation of resident cells by AIF-1 overexpression.
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http://dx.doi.org/10.1007/s00395-013-0368-7DOI Listing
July 2013

Muscle type and fiber type specificity in muscle wasting.

Int J Biochem Cell Biol 2013 Oct 21;45(10):2191-9. Epub 2013 May 21.

Venetian Institute of Molecular Medicine VIMM, Padova, Italy.

Muscle wasting occurs in a variety of conditions, including both genetic diseases, such as muscular dystrophies, and acquired disorders, ranging from muscle disuse to cancer cachexia, from heart failure to aging sarcopenia. In most of these conditions, the loss of muscle tissue is not homogeneous, but involves specific muscle groups, for example Duchenne muscular dystrophy affects most body muscles but spares extraocular muscles, and other dystrophies affect selectively proximal or distal limb muscles. In addition, muscle atrophy can affect specific fiber types, involving predominantly slow type 1 or fast type 2 muscle fibers, and is frequently accompanied by a slow-to-fast or fast-to-slow fiber type shift. For example, muscle disuse, such as spinal cord injury, causes type 1 fiber atrophy with a slow-to-fast fiber type shift, whereas cancer cachexia leads to preferential atrophy of type 2 fibers with a fast-to-slow fiber type shift. The identification of the signaling pathways responsible for the differential response of muscles types and fiber types can lead to a better understanding of the pathogenesis of muscle wasting and to the design of therapeutic interventions appropriate for the specific disorders. This article is part of a Directed Issue entitled: Molecular basis of muscle wasting.
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http://dx.doi.org/10.1016/j.biocel.2013.05.016DOI Listing
October 2013

Mechanisms regulating skeletal muscle growth and atrophy.

FEBS J 2013 Sep 17;280(17):4294-314. Epub 2013 Apr 17.

Venetian Institute of Molecular Medicine, Padova, Italy.

Skeletal muscle mass increases during postnatal development through a process of hypertrophy, i.e. enlargement of individual muscle fibers, and a similar process may be induced in adult skeletal muscle in response to contractile activity, such as strength exercise, and specific hormones, such as androgens and β-adrenergic agonists. Muscle hypertrophy occurs when the overall rates of protein synthesis exceed the rates of protein degradation. Two major signaling pathways control protein synthesis, the IGF1-Akt-mTOR pathway, acting as a positive regulator, and the myostatin-Smad2/3 pathway, acting as a negative regulator, and additional pathways have recently been identified. Proliferation and fusion of satellite cells, leading to an increase in the number of myonuclei, may also contribute to muscle growth during early but not late stages of postnatal development and in some forms of muscle hypertrophy in the adult. Muscle atrophy occurs when protein degradation rates exceed protein synthesis, and may be induced in adult skeletal muscle in a variety of conditions, including starvation, denervation, cancer cachexia, heart failure and aging. Two major protein degradation pathways, the proteasomal and the autophagic-lysosomal pathways, are activated during muscle atrophy and variably contribute to the loss of muscle mass. These pathways involve a variety of atrophy-related genes or atrogenes, which are controlled by specific transcription factors, such as FoxO3, which is negatively regulated by Akt, and NF-κB, which is activated by inflammatory cytokines.
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http://dx.doi.org/10.1111/febs.12253DOI Listing
September 2013

Adaptation of mouse skeletal muscle to long-term microgravity in the MDS mission.

PLoS One 2012 28;7(3):e33232. Epub 2012 Mar 28.

Department of Biomedical Sciences, University of Padova, Padova, Italy.

The effect of microgravity on skeletal muscles has so far been examined in rat and mice only after short-term (5-20 day) spaceflights. The mice drawer system (MDS) program, sponsored by Italian Space Agency, for the first time aimed to investigate the consequences of long-term (91 days) exposure to microgravity in mice within the International Space Station. Muscle atrophy was present indistinctly in all fiber types of the slow-twitch soleus muscle, but was only slightly greater than that observed after 20 days of spaceflight. Myosin heavy chain analysis indicated a concomitant slow-to-fast transition of soleus. In addition, spaceflight induced translocation of sarcolemmal nitric oxide synthase-1 (NOS1) into the cytosol in soleus but not in the fast-twitch extensor digitorum longus (EDL) muscle. Most of the sarcolemmal ion channel subunits were up-regulated, more in soleus than EDL, whereas Ca(2+)-activated K(+) channels were down-regulated, consistent with the phenotype transition. Gene expression of the atrophy-related ubiquitin-ligases was up-regulated in both spaceflown soleus and EDL muscles, whereas autophagy genes were in the control range. Muscle-specific IGF-1 and interleukin-6 were down-regulated in soleus but up-regulated in EDL. Also, various stress-related genes were up-regulated in spaceflown EDL, not in soleus. Altogether, these results suggest that EDL muscle may resist to microgravity-induced atrophy by activating compensatory and protective pathways. Our study shows the extended sensitivity of antigravity soleus muscle after prolonged exposition to microgravity, suggests possible mechanisms accounting for the resistance of EDL, and individuates some molecular targets for the development of countermeasures.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0033232PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3314659PMC
August 2012

Regeneration of mammalian skeletal muscle. Basic mechanisms and clinical implications.

Curr Pharm Des 2010 ;16(8):906-14

Venetian Institute of Molecular Medicine, Via Orus 2, 35129 Padova, Italy.

Mammalian skeletal muscles can regenerate following injury and this response is mediated by a specific type of stem cell, the satellite cell. We review here the three main phases of muscle regeneration, including i) the initial inflammatory response and the dual role of macrophages as both scavengers involved in the phagocytosis of necrotic debris and promoters of myogenic differentiation, ii) the activation and differentiation of satellite cells and iii) the growth and remodeling of the regenerated muscle tissue. Nerve activity is required to support the growth of regenerated myofibers and the specification of muscle fiber types, in particular the activation of the slow gene program. We discuss the regeneration process in two different settings. Chronic degenerative diseases, such as muscular dystrophies, are characterized by repeated cycles of segmental necrosis and regeneration involving scattered myofibers. In these conditions the regenerative capacity of satellite cells becomes exhausted with time and fibrosis prevails. Acute traumatic injuries, such as strain injuries common in sport medicine, cause the rupture of large myofiber bundles leading to muscle regeneration and formation of scar tissue and new myotendinous junctions at the level of the rupture. Mechanical loading is essential for muscle regeneration, therefore, following initial immobilization to avoid the risk of reruptures, early remobilization is required to induce correct growth and orientation of regenerated myofibers. Finally, we discuss the causes of age-dependent decline in muscle regeneration potential and the possibility of boosting regeneration in aging muscle and in muscular dystrophies.
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http://dx.doi.org/10.2174/138161210790883453DOI Listing
June 2010

NFAT isoforms control activity-dependent muscle fiber type specification.

Proc Natl Acad Sci U S A 2009 Aug 24;106(32):13335-40. Epub 2009 Jul 24.

Department of Biomedical Sciences, University of Padova, 35131 Padova, Italy.

The intracellular signals that convert fast and slow motor neuron activity into muscle fiber type specific transcriptional programs have only been partially defined. The calcium/calmodulin-dependent phosphatase calcineurin (Cn) has been shown to mediate the transcriptional effects of motor neuron activity, but precisely how 4 distinct muscle fiber types are composed and maintained in response to activity is largely unknown. Here, we show that 4 nuclear factor of activated T cell (NFAT) family members act coordinately downstream of Cn in the specification of muscle fiber types. We analyzed the role of NFAT family members in vivo by transient transfection in skeletal muscle using a loss-of-function approach by RNAi. Our results show that, depending on the applied activity pattern, different combinations of NFAT family members translocate to the nucleus contributing to the transcription of fiber type specific genes. We provide evidence that the transcription of slow and fast myosin heavy chain (MyHC) genes uses different combinations of NFAT family members, ranging from MyHC-slow, which uses all 4 NFAT isoforms, to MyHC-2B, which only uses NFATc4. Our data contribute to the elucidation of the mechanisms whereby activity can modulate the phenotype and performance of skeletal muscle.
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http://dx.doi.org/10.1073/pnas.0812911106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2726382PMC
August 2009