Publications by authors named "Enrico Pierantozzi"

20 Publications

  • Page 1 of 1

Calsequestrin, a key protein in striated muscle health and disease.

J Muscle Res Cell Motil 2020 Jun 2. Epub 2020 Jun 2.

Molecular Medicine Section, Department of Molecular and Developmental Medicine, University of Siena, Via A. Moro, 2, 53100, Siena, Italy.

Calsequestrin (CASQ) is the most abundant Ca binding protein localized in the sarcoplasmic reticulum (SR) of skeletal and cardiac muscle. The genome of vertebrates contains two genes, CASQ1 and CASQ2. CASQ1 and CASQ2 have a high level of homology, but show specific patterns of expression. Fast-twitch skeletal muscle fibers express only CASQ1, both CASQ1 and CASQ2 are present in slow-twitch skeletal muscle fibers, while CASQ2 is the only protein present in cardiomyocytes. Depending on the intraluminal SR Ca levels, CASQ monomers assemble to form large polymers, which increase their Ca binding ability. CASQ interacts with triadin and junctin, two additional SR proteins which contribute to localize CASQ to the junctional region of the SR (j-SR) and also modulate CASQ ability to polymerize into large macromolecular complexes. In addition to its ability to bind Ca in the SR, CASQ appears also to be able to contribute to regulation of Ca homeostasis in muscle cells. Both CASQ1 and CASQ2 are able to either activate and inhibit the ryanodine receptors (RyRs) calcium release channels, likely through their interactions with junctin and triadin. Additional evidence indicates that CASQ1 contributes to regulate the mechanism of store operated calcium entry in skeletal muscle via a direct interaction with the Stromal Interaction Molecule 1 (STIM1). Mutations in CASQ2 and CASQ1 have been identified, respectively, in patients with catecholamine-induced polymorphic ventricular tachycardia and in patients with some forms of myopathy. This review will highlight recent developments in understanding CASQ1 and CASQ2 in health and diseases.
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http://dx.doi.org/10.1007/s10974-020-09583-6DOI Listing
June 2020

A novel homozygous mutation in the TRDN gene causes a severe form of pediatric malignant ventricular arrhythmia.

Heart Rhythm 2020 02 19;17(2):296-304. Epub 2019 Aug 19.

Vita-Salute San Raffaele University, Milan, Italy; Genomic Unit for the Diagnosis of Human Pathologies, Division of Genetics and Cellular Biology, IRCCS San Raffaele Hospital, Milan, Italy. Electronic address:

Background: Triadin is a protein expressed in cardiac and skeletal muscle that has an essential role in the structure and functional regulation of calcium release units and excitation-contraction coupling. Mutations in the triadin gene (TRDN) have been described in different forms of human arrhythmia syndromes with early onset and severe arrhythmogenic phenotype, including triadin knockout syndrome.

Objective: The purpose of this study was to characterize the pathogenetic mechanism underlying a case of severe pediatric malignant arrhythmia associated with a defect in the TRDN gene.

Methods: We used a trio whole exome sequencing approach to identify the genetic defect in a 2-year-old boy who had been resuscitated from sudden cardiac arrest and had frequent episodes of ventricular fibrillation and a family history positive for sudden death. We then performed in vitro functional analysis to investigate possible pathogenic mechanisms underlying this severe phenotype.

Results: We identified a novel homozygous missense variant (p.L56P) in the TRDN gene in the proband that was inherited from the heterozygous unaffected parents. Expression of a green fluorescent protein (GFP)-tagged mutant human cardiac triadin isoform (TRISK32-L56P-GFP) in heterologous systems revealed that the mutation alters protein dynamics. Furthermore, when co-expressed with the type 2 ryanodine receptor, caffeine-induced calcium release from TRISK32-L56P-GFP was relatively lower compared to that observed with the wild-type construct.

Conclusion: The results of this study allowed us to hypothesize a pathogenic mechanism underlying this rare arrhythmogenic recessive form, suggesting that the mutant protein potentially can trigger arrhythmias by altering calcium homeostasis.
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http://dx.doi.org/10.1016/j.hrthm.2019.08.018DOI Listing
February 2020

Calcium Homeostasis Is Modified in Skeletal Muscle Fibers of Small Ankyrin1 Knockout Mice.

Int J Mol Sci 2019 Jul 9;20(13). Epub 2019 Jul 9.

Department of Physiology, Medical Faculty, University of Debrecen, H-4002 Debrecen, Hungary.

Small Ankyrins (sAnk1) are muscle-specific isoforms generated by the gene that participate in the organization of the sarcoplasmic reticulum (SR) of striated muscles. Accordingly, the volume of SR tubules localized around the myofibrils is strongly reduced in skeletal muscle fibers of 4- and 10-month-old sAnk1 knockout (KO) mice, while additional structural alterations only develop with aging. To verify whether the lack of sAnk1 also alters intracellular Ca handling, cytosolic Ca levels were analyzed in stimulated skeletal muscle fibers from 4- and 10-month-old sAnk1 KO mice. The SR Ca content was reduced in sAnk1 KO mice regardless of age. The amplitude of the Ca transients induced by depolarizing pulses was decreased in myofibers of sAnk1 KO with respect to wild type (WT) fibers, while their voltage dependence was not affected. Furthermore, analysis of spontaneous Ca release events (sparks) on saponin-permeabilized muscle fibers indicated that the frequency of sparks was significantly lower in fibers from 4-month-old KO mice compared to WT. Furthermore, both the amplitude and spatial spread of sparks were significantly smaller in muscle fibers from both 4- and 10-month-old KO mice compared to WT. These data suggest that the absence of sAnk1 results in an impairment of SR Ca release, likely as a consequence of a decreased Ca store due to the reduction of the SR volume in sAnk1 KO muscle fibers.
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http://dx.doi.org/10.3390/ijms20133361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6651408PMC
July 2019

Molecular determinants of homo- and heteromeric interactions of Junctophilin-1 at triads in adult skeletal muscle fibers.

Proc Natl Acad Sci U S A 2019 07 17;116(31):15716-15724. Epub 2019 Jul 17.

Department of Molecular and Developmental Medicine, Molecular Medicine Section, University of Siena, 53100 Siena, Italy;

In adult skeletal muscles, 2 junctophilin isoforms (JPH1 and JPH2) tether the sarcoplasmic reticulum (SR) to transverse tubule (T-tubule) membranes, generating stable membrane contact sites known as triads. JPHs are anchored to the membrane of the SR by a C-terminal transmembrane domain (TMD) and bind the T-tubule membrane through their cytosolic N-terminal region, which contains 8 lipid-binding (MORN) motifs. By combining expression of GFP-JPH1 deletion mutants in skeletal muscle fibers with in vitro biochemical experiments, we investigated the molecular determinants of JPH1 recruitment at triads in adult skeletal muscle fibers. We found that MORN motifs bind PI(4,5)P in the sarcolemma, but do not mediate the selective localization of JPH1 at the T-tubule compartment of triads. On the contrary, fusion proteins containing only the TMD of JPH1 were able to localize at the junctional SR compartment of the triad. Bimolecular fluorescence complementation experiments indicated that the TMD of JPH1 can form dimers, suggesting that the observed localization at triads may result from dimerization with the TMDs of resident JPH1. A second domain, capable of mediating homo- and heterodimeric interactions between JPH1 and JPH2 was identified in the cytosolic region. FRAP experiments revealed that removal of either one of these 2 domains in JPH1 decreases the association of the resulting mutant proteins with triads. Altogether, these results suggest that the ability to establish homo- and heterodimeric interactions with resident JPHs may support the recruitment and stability of newly synthesized JPHs at triads in adult skeletal muscle fibers.
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http://dx.doi.org/10.1073/pnas.1820980116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6681751PMC
July 2019

Functional Electrical Stimulation: A Possible Strategy to Improve Muscle Function in Central Core Disease?

Front Neurol 2019 29;10:479. Epub 2019 May 29.

CeSI-Met-Center for Research on Ageing and Translational Medicine, University G. d'Annunzio, Chieti, Italy.

Central Core Disease (CCD) is a congenital myopathy characterized by presence of amorphous central areas (or ) lacking glycolytic/oxidative enzymes and mitochondria in skeletal muscle fibers. Most CCD families are linked to mutations in ryanodine receptor type-1 (RYR1), the gene encoding for the sarcoplasmic reticulum (SR) Ca release channel of skeletal muscle. As no treatments are available for CCD, currently management of patients is essentially based on a physiotherapic approaches. Functional electrical stimulation (FES) is a technique used to deliver low energy electrical impulses to artificially stimulate selected skeletal muscle groups. Here we tested the efficacy of FES in counteracting muscle loss and improve function in the lower extremities of a 55-year-old female patient which was diagnosed with CCD at the age of 44. Genetic screening of the RyR1 gene identified a missense mutation (c.7354C>T) in exon 46 resulting in an amino acid substitution (p.R2452W) and a duplication (c.12853_12864dup12) in exon 91. The patient was treated with FES for 26 months and subjected before, during, and after training to a series of functional and structural assessments: measurement of maximum isometric force of leg extensor muscles, magnetic resonance imaging, a complete set of functional tests to assess mobility in activities of daily living, and analysis of muscle biopsies by histology and electron microscopy. All results point to an improvement in muscle structure and function induced by FES suggesting that this approach could be considered as an additional supportive measure to maintain/improve muscle function (and possibly reduce muscle loss) in CCD patients.
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http://dx.doi.org/10.3389/fneur.2019.00479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548841PMC
May 2019

Murine obscurin and Obsl1 have functionally redundant roles in sarcolemmal integrity, sarcoplasmic reticulum organization, and muscle metabolism.

Commun Biol 2019 9;2:178. Epub 2019 May 9.

1Division of Cardiology, School of Medicine, University of California, San Diego, 92093 CA USA.

Biological roles of obscurin and its close homolog Obsl1 (obscurin-like 1) have been enigmatic. While obscurin is highly expressed in striated muscles, Obsl1 is found ubiquitously. Accordingly, obscurin mutations have been linked to myopathies, whereas mutations in Obsl1 result in 3M-growth syndrome. To further study unique and redundant functions of these closely related proteins, we generated and characterized Obsl1 knockouts. Global Obsl1 knockouts are embryonically lethal. In contrast, skeletal muscle-specific Obsl1 knockouts show a benign phenotype similar to obscurin knockouts. Only deletion of both proteins and removal of their functional redundancy revealed their roles for sarcolemmal stability and sarcoplasmic reticulum organization. To gain unbiased insights into changes to the muscle proteome, we analyzed tibialis anterior and soleus muscles by mass spectrometry, uncovering additional changes to the muscle metabolism. Our analyses suggest that all obscurin protein family members play functions for muscle membrane systems.
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http://dx.doi.org/10.1038/s42003-019-0405-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6509138PMC
April 2020

Putative endothelial progenitor cells predict long-term mortality in type-2 diabetes.

Endocrine 2018 10 30;62(1):263-266. Epub 2018 Jul 30.

Department of Molecular and Developmental Medicine, Molecular Medicine Section, University of Siena, Siena, Italy.

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http://dx.doi.org/10.1007/s12020-018-1695-0DOI Listing
October 2018

Mesenchymal stem cells: from the perivascular environment to clinical applications.

Histol Histopathol 2018 Dec 7;33(12):1235-1246. Epub 2018 May 7.

Molecular Medicine Section, Department of Molecular and Developmental Medicine, University of Siena, Siena, Italy.

Adult stem cells represent a fundamental biological system that has fascinated scientists over the last decades, and are currently the subject of a large number of studies aimed at better defining the properties of these cells, with a prominent focus on improving their application in regenerative medicine. One of the most used adult stem cells in clinical trials are mesenchymal stem cells (MSCs), which are multipotent cells able to differentiate into mature cells of mesodermal lineages. Following the initial studies on MSCs isolated from bone marrow, similar cells were also isolated from a variety of fetal and adult human tissues. Initially considered as identical and equipotent, MSCs from tissues other than bone marrow actually display differences in terms of their plastic abilities, which can be ascribed to the tissue of origin and/or to the procedures used for their isolation. Moreover, results from additional studies suggest that cultured MSCs represent the in vitro version of a subset of in vivo resident cells localized in the perivascular environment. In this review, we will focus our attention on MSCs from tissues other than bone marrow, their in vivo localization and their current applications in clinics.
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http://dx.doi.org/10.14670/HH-11-998DOI Listing
December 2018

A novel FLNC frameshift and an OBSCN variant in a family with distal muscular dystrophy.

PLoS One 2017 26;12(10):e0186642. Epub 2017 Oct 26.

Molecular Medicine Section, Department of Molecular and Developmental Medicine, University of Siena and Azienda Ospedaliera Universitaria Senese, Siena, Italy.

A novel FLNC c.5161delG (p.Gly1722ValfsTer61) mutation was identified in two members of a French family affected by distal myopathy and in one healthy relative. This FLNC c.5161delG mutation is one nucleotide away from a previously reported FLNC mutation (c.5160delC) that was identified in patients and in asymptomatic carriers of three Bulgarian families with distal muscular dystrophy, indicating a low penetrance of the FLNC frameshift mutations. Given these similarities, we believe that the two FLNC mutations alone can be causative of distal myopathy without full penetrance. Moreover, comparative analysis of the clinical manifestations indicates that patients of the French family show an earlier onset and a complete segregation of the disease. As a possible explanation of this, the two French patients also carry a OBSCN c.13330C>T (p.Arg4444Trp) mutation. The p.Arg4444Trp variant is localized within the OBSCN Ig59 domain that, together with Ig58, binds to the ZIg9/ZIg10 domains of titin at Z-disks. Structural and functional studies indicate that this OBSCN p.Arg4444Trp mutation decreases titin binding by ~15-fold. On this line, we suggest that the combination of the OBSCN p.Arg4444Trp variant and of the FLNC c.5161delG mutation, can cooperatively affect myofibril stability and increase the penetrance of muscular dystrophy in the French family.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0186642PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5657976PMC
November 2017

Identification and characterization of three novel mutations in the CASQ1 gene in four patients with tubular aggregate myopathy.

Hum Mutat 2017 12 26;38(12):1761-1773. Epub 2017 Sep 26.

Department of Molecular and Developmental Medicine, Molecular Medicine Section, University of Siena, Siena, Italy.

Here, we report the identification of three novel missense mutations in the calsequestrin-1 (CASQ1) gene in four patients with tubular aggregate myopathy. These CASQ1 mutations affect conserved amino acids in position 44 (p.(Asp44Asn)), 103 (p.(Gly103Asp)), and 385 (p.(Ile385Thr)). Functional studies, based on turbidity and dynamic light scattering measurements at increasing Ca concentrations, showed a reduced Ca -dependent aggregation for the CASQ1 protein containing p.Asp44Asn and p.Gly103Asp mutations and a slight increase in Ca -dependent aggregation for the p.Ile385Thr. Accordingly, limited trypsin proteolysis assay showed that p.Asp44Asn and p.Gly103Asp were more susceptible to trypsin cleavage in the presence of Ca in comparison with WT and p.Ile385Thr. Analysis of single muscle fibers of a patient carrying the p.Gly103Asp mutation showed a significant reduction in response to caffeine stimulation, compared with normal control fibers. Expression of CASQ1 mutations in eukaryotic cells revealed a reduced ability of all these CASQ1 mutants to store Ca and a reduced inhibitory effect of p.Ile385Thr and p.Asp44Asn on store operated Ca entry. These results widen the spectrum of skeletal muscle diseases associated with CASQ1 and indicate that these mutations affect properties critical for correct Ca handling in skeletal muscle fibers.
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http://dx.doi.org/10.1002/humu.23338DOI Listing
December 2017

The potential of obscurin as a therapeutic target in muscle disorders.

Expert Opin Ther Targets 2017 09 4;21(9):897-910. Epub 2017 Aug 4.

b Molecular Medicine Section, Department of Molecular and Developmental Medicine , University of Siena , Siena , Italy.

Introduction: Obscurin, a giant protein of striated muscles, is emerging as an important player in a wide range of processes including myofibril assembly and maintenance, muscle protein degradation and intracellular signaling. Accordingly, obscurin participates to the mechanisms by which muscles adapt to physiological requirements or to pathological cues associated with cardiac and skeletal muscle diseases. Areas covered: The structure of the different obscurin isoforms identified so far, their tissue distribution and the most recent findings on obscurin in invertebrates and mammals will be reviewed. We will provide a synopsis of known molecular interactions between obscurin and other proteins and the biological relevance of these interactions for striated muscle function. The involvement of obscurin in protein degradation mechanisms and intracellular signaling will be also discussed along with initial evidence of a role of obscurin in the pathophysiology of human diseases. Expert opinion: Although still much remains to be discovered about the role of obscurin either as a structural component of the sarcomere or as a mediator of signaling pathways within muscle cells, it can be envisioned that this protein represents an interesting novel pharmacological target for the prevention and treatment of cardiac and skeletal muscle diseases.
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http://dx.doi.org/10.1080/14728222.2017.1361931DOI Listing
September 2017

Not All Pericytes Are Born Equal: Pericytes from Human Adult Tissues Present Different Differentiation Properties.

Stem Cells Dev 2016 10 22;25(20):1549-1558. Epub 2016 Aug 22.

Molecular Medicine Section, Department of Molecular and Developmental Medicine, University of Siena , Siena, Italy .

Pericytes (PCs) have been recognized for a long time only as structural cells of the blood vessels. The identification of tight contacts with endothelial cells and the ability to interact with surrounding cells through paracrine signaling revealed additional functions of PCs in maintaining the homeostasis of the perivascular environment. PCs got the front page, in the late 1990s, after the identification and characterization of a new embryonic cell population, the mesoangioblasts, from which PCs present in the adult organism are thought to derive. From these studies, it was clear that PCs were also endowed with multipotent mesodermal abilities. Furthermore, their ability to cross the vascular wall and to reconstitute skeletal muscle tissue after systemic injection opened the way to a number of studies aimed to develop therapeutic protocols for a cell therapy of muscular dystrophy. This has resulted in a major effort to characterize pericytic cell populations from skeletal muscle and other adult tissues. Additional studies also addressed their relationship with other cells of the perivascular compartment and with mesenchymal stem cells. These data have provided initial evidence that PCs from different adult tissues might be endowed with distinctive differentiation abilities. This would suggest that the multipotent mesenchymal ability of PCs might be restrained within different tissues, likely depending on the specific cell renewal and repair requirements of each tissue. This review presents current knowledge on human PCs and highlights recent data on the differentiation properties of PCs isolated from different adult tissues.
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http://dx.doi.org/10.1089/scd.2016.0177DOI Listing
October 2016

Tissue-Specific Cultured Human Pericytes: Perivascular Cells from Smooth Muscle Tissue Have Restricted Mesodermal Differentiation Ability.

Stem Cells Dev 2016 05 8;25(9):674-86. Epub 2016 Apr 8.

1 Molecular Medicine Section, University of Siena , Siena, Italy .

Microvascular pericytes (PCs) are considered the adult counterpart of the embryonic mesoangioblasts, which represent a multipotent cell population that resides in the dorsal aorta of the developing embryo. Although PCs have been isolated from several adult organs and tissues, it is still controversial whether PCs from different tissues exhibit distinct differentiation potentials. To address this point, we investigated the differentiation potentials of isogenic human cultured PCs isolated from skeletal (sk-hPCs) and smooth muscle tissues (sm-hPCs). We found that both sk-hPCs and sm-hPCs expressed known pericytic markers and did not express endothelial, hematopoietic, and myogenic markers. Both sk-hPCs and sm-hPCs were able to differentiate into smooth muscle cells. In contrast, sk-hPCs, but not sm-hPCs, differentiated in skeletal muscle cells and osteocytes. Given the reported ability of the Notch pathway to regulate skeletal muscle and osteogenic differentiation, sk-hPCs and sm-hPCs were treated with N-[N-(3,5- difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), a known inhibitor of Notch signaling. DAPT treatment, as assessed by histological and molecular analysis, enhanced myogenic differentiation and abolished osteogenic potential of sk-hPCs. In contrast, DAPT treatment did not affect either myogenic or osteogenic differentiation of sm-hPCs. In summary, these results indicate that, despite being isolated from the same anatomical niche, cultured PCs from skeletal muscle and smooth muscle tissues display distinct differentiation abilities.
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http://dx.doi.org/10.1089/scd.2015.0336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4854214PMC
May 2016

A mutation in the CASQ1 gene causes a vacuolar myopathy with accumulation of sarcoplasmic reticulum protein aggregates.

Hum Mutat 2014 Oct 10;35(10):1163-70. Epub 2014 Sep 10.

Molecular Medicine Section, Department of Molecular and Developmental Medicine, University of Siena and Azienda Ospedaliera Universitaria Senese, Siena, 53100, Italy; IIM, Interuniversity Institute of Myology.

A missense mutation in the calsequestrin-1 gene (CASQ1) was found in a group of patients with a myopathy characterized by weakness, fatigue, and the presence of large vacuoles containing characteristic inclusions resulting from the aggregation of sarcoplasmic reticulum (SR) proteins. The mutation affects a conserved aspartic acid in position 244 (p.Asp244Gly) located in one of the high-affinity Ca(2+) -binding sites of CASQ1 and alters the kinetics of Ca(2+) release in muscle fibers. Expression of the mutated CASQ1 protein in COS-7 cells showed a markedly reduced ability in forming elongated polymers, whereas both in cultured myotubes and in in vivo mouse fibers induced the formation of electron-dense SR vacuoles containing aggregates of the mutant CASQ1 protein that resemble those observed in muscle biopsies of patients. Altogether, these results support the view that a single missense mutation in the CASQ1 gene causes the formation of abnormal SR vacuoles containing aggregates of CASQ1, and other SR proteins, results in altered Ca(2+) release in skeletal muscle fibers, and, hence, is responsible for the clinical phenotype observed in these patients.
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http://dx.doi.org/10.1002/humu.22631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4177304PMC
October 2014

Distinct regions of triadin are required for targeting and retention at the junctional domain of the sarcoplasmic reticulum.

Biochem J 2014 Mar;458(2):407-17

*Department of Molecular and Developmental Medicine, University of Siena, Via A Moro 2, 53100 Siena, Italy.

Ca2+ release, which is necessary for muscle contraction, occurs at the j-SR (junctional domain of the sarcoplasmic reticulum). It requires the assembly of a large multiprotein complex containing the RyR (ryanodine receptor) and additional proteins, including triadin and calsequestrin. The signals which drive these proteins to the j-SR and how they assemble to form this multiprotein complex are poorly understood. To address aspects of these questions we studied the localization, dynamic properties and molecular interactions of triadin. We identified three regions, named TR1 (targeting region 1), TR2 and TR3, that contribute to the localization of triadin at the j-SR. FRAP experiments showed that triadin is stably associated with the j-SR and that this association is mediated by TR3. Protein pull-down experiments indicated that TR3 contains binding sites for calsequestrin-1 and that triadin clustering can be enhanced by binding to calsequestrin-1. These findings were confirmed by FRET experiments. Interestingly, the stable association of triadin to the j-SR was significantly decreased in myotubes from calsequestrin-1 knockout mice. Taken together, these results identify three regions in triadin that mediate targeting to the j-SR and reveal a role for calsequestrin-1 in promoting the stable association of triadin to the multiprotein complex associated with RyR.
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http://dx.doi.org/10.1042/BJ20130719DOI Listing
March 2014

Obscurin is required for ankyrinB-dependent dystrophin localization and sarcolemma integrity.

J Cell Biol 2013 Feb;200(4):523-36

Molecular Medicine Section, Department of Molecular and Developmental Medicine, University of Siena, 53100 Siena, Italy.

Obscurin is a large myofibrillar protein that contains several interacting modules, one of which mediates binding to muscle-specific ankyrins. Interaction between obscurin and the muscle-specific ankyrin sAnk1.5 regulates the organization of the sarcoplasmic reticulum in striated muscles. Additional muscle-specific ankyrin isoforms, ankB and ankG, are localized at the subsarcolemma level, at which they contribute to the organization of dystrophin and β-dystroglycan at costameres. In this paper, we report that in mice deficient for obscurin, ankB was displaced from its localization at the M band, whereas localization of ankG at the Z disk was not affected. In obscurin KO mice, localization at costameres of dystrophin, but not of β-dystroglycan, was altered, and the subsarcolemma microtubule cytoskeleton was disrupted. In addition, these mutant mice displayed marked sarcolemmal fragility and reduced muscle exercise tolerance. Altogether, the results support a model in which obscurin, by targeting ankB at the M band, contributes to the organization of subsarcolemma microtubules, localization of dystrophin at costameres, and maintenance of sarcolemmal integrity.
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http://dx.doi.org/10.1083/jcb.201205118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3575540PMC
February 2013

Multi-potent progenitors in freshly isolated and cultured human mesenchymal stem cells: a comparison between adipose and dermal tissue.

Cell Tissue Res 2011 Apr 19;344(1):85-95. Epub 2011 Feb 19.

Molecular Medicine Section, Department of Neuroscience, and Center for Stem Cell Research, University of Siena, Via Aldo Moro, 53100, Siena, Italy.

Mesenchymal stem cells (MSCs) from human adult adipose tissue (A-MSCs) have a better differentiative ability than MSCs derived from the derma (D-MSCs). To test whether this difference is associated with differences in the content of multi-potent progenitors in A-MSCs, the number and the differentiative properties of multi-potent progenitors have been analyzed in various preparations of A-MSCs and D-MSCs. Adipogenic and osteogenic differentiation performed on colony-forming units have revealed that adipogenic and osteogenic progenitors are similar in the two populations, with only a slighty better performance of A-MSCs over D-MSCs from passages p0 to p15. An analysis of the presence of tri-, bi-, uni- and nulli-potent progenitors isolated immediately after isolation from tissues (p0) has shown comparable numbers of tri-potent and bi-potent progenitors in MSCs from the two tissues, whereas a higher content in uni-potent cells committed to adipocytes and a lower content in nulli-potent cells has been observed in A-MSCs. Furthermore, we have characterized the progenitors present in A-MSCs after six passages in vitro to verify the way in which in vitro culture can affect content in progenitor cells. We have observed that the percentage of tri-potent cells in A-MSCs at p6 remains similar to that observed at p0, although bi-potent and uni-potent progenitors committed to osteogenic differentiation increase at p6, whereas nulli-potent cells decrease at p6. These data indicate that the greater differentiative ability of A-MSC populations does not correlate directly with the number of multi-potent progenitors, suggesting that other factors influence the differentiation of bulk populations of A-MSCs.
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http://dx.doi.org/10.1007/s00441-011-1139-0DOI Listing
April 2011

Pluripotency regulators in human mesenchymal stem cells: expression of NANOG but not of OCT-4 and SOX-2.

Stem Cells Dev 2011 May 29;20(5):915-23. Epub 2010 Oct 29.

Molecular Medicine Section, Department of Neuroscience, University of Siena, Siena, Italy.

Mesenchymal stem cells (MSCs) are adult multipotent cells able to differentiate toward mature mesodermal lineages. In spite of more than a decade of investigation, little is known about the molecular mechanisms regulating the undifferentiated state and the identity of distinct functional subpopulations in these cells. Transcription factors that regulate the maintenance of the pluripotent state in embryonic stem cells, including NANOG, SOX2, and OCT4, have been proposed to play a similar role also in adult stem cells, although with conflicting results. We performed a critical evaluation of expression of these 3 transcription factors and found that NANOG, but not OCT-4 and SOX-2, is expressed in cultured human adult MSCs. Actually, NANOG was not expressed in freshly isolated MSCs, but was detected only after in vitro culture. NANOG was detected only in proliferating cells, but not in MSCs induced to differentiate. The percentage of cells expressing NANOG was maintained throughout early passages of MSCs, but then started to decrease in late passages in MSCs from adipose tissue and heart but not from bone marrow. However, the number of NANOG-expressing cells did not associate with the proliferative and differentiative capabilities of MSC populations, neither its expression appeared to identify cells having stem or progenitor cell properties. Accordingly, we propose that activation of NANOG expression in MSCs is associated with, although cannot directly regulate, the transition from in vivo quiescence to adaptation to in vitro growth conditions.
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http://dx.doi.org/10.1089/scd.2010.0353DOI Listing
May 2011

p75 neurotrophin receptor is involved in proliferation of undifferentiated mouse embryonic stem cells.

Exp Cell Res 2009 Nov 28;315(18):3220-32. Epub 2009 Aug 28.

Department of Public Health and Cell Biology, Section of Histology and Embryology, University of Rome Tor Vergata, Via Montpellier 1, 00133 Rome, Italy.

Neurotrophins and their receptors are known to play a role in the proliferation and survival of many different cell types of neuronal and non-neuronal lineages. In addition, there is much evidence in the literature showing that the p75 neurotrophin receptor (p75(NTR)), alone or in association with members of the family of Trk receptors, is expressed in a wide variety of stem cells, although its role in such cells has not been completely elucidated. In the present work we have investigated the expression of p75(NTR) and Trks in totipotent and pluripotent cells, the mouse pre-implantation embryo and embryonic stem and germ cells (ES and EG cells). p75(NTR) and TrkA can be first detected in the blastocyst from which ES cell lines are derived. Mouse ES cells retain p75(NTR)/TrkA expression. Nerve growth factor is the only neurotrophin able to stimulate ES cell growth in culture, without affecting the expression of stem cell markers, alkaline phosphatase, Oct4 and Nanog. Such proliferation effect was blocked by antagonizing either p75(NTR) or TrkA. Interestingly, immunoreactivity to anti-p75(NTR) antibodies is lost upon ES cell differentiation. The expression pattern of neurotrophin receptors in murine ES cells differs from human ES cells, that only express TrkB and C, and do not respond to NGF. In this paper we also show that, while primordial germ cells (PGC) do not express p75(NTR), when they are made to revert to an ES-like phenotype, becoming EG cells, expression of p75(NTR) is turned on.
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http://dx.doi.org/10.1016/j.yexcr.2009.08.014DOI Listing
November 2009

Thyroid status affects rat liver regeneration after partial hepatectomy by regulating cell cycle and apoptosis.

Cell Physiol Biochem 2005 ;15(1-4):69-76

Department of Cellular and Developmental Biology, University La Sapienza, Rome, Italy.

In rats, various growth factors and hormones, as well as partial hepatectomy (PH) are able to trigger the proliferative response of hepatocytes. Although recent evidence highlights the important role of thyroid hormones and thyroid status in regulating the growth of liver cells in vitro and in vivo models, the mechanism involved in the pro-proliferative effects of thyroid hormones is still unclear. Here we have investigated how in rats made hypo- and hyperthyroid after prolonged treatment respectively with propylthiouracil (PTU) and triiodothyronine (T3), the thyroid status affects liver regeneration after PH by regulating cell cycle and apoptosis proteins. Our results show that both in control and partially hepatectomized animals hyperthyroidism increases the cyclin D1, E and A levels and the activity of cyclin-cdk complexes, and decreases the levels of cdk inhibitors such as p16 and p27. On the contrary hypothyroidism induces a down-regulation of the activity of cyclin cdk complexes decreasing cyclin levels. Thyroid hormones control also p53 and p73, two proteins involved in apoptosis and growth arrest which are induced by PH. In particular, hypothyroidism increases and T3 treatment decreases p73 levels. The analysis of the phosphorylated forms of p42/44 and p38 MAPK revealed that they are induced during hepatic regeneration in euthyroid and hyperthyroid rats whereas they are negatively regulated in hypothyroid rats. In conclusion our data demonstrate that thyroid status can affects liver regeneration, altering the expression and the activity of the proteins involved in the control of cell cycle and growth arrest.
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http://dx.doi.org/10.1159/000083639DOI Listing
August 2005