Enriching Satellite Cells with α2δ1 Promotes Differentiation
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1847-Pos Board B617 Different Capacity for Store-Operated Ca 2D Entry and Ca 2D Extrusion Across the Plasma Membrane of Wild-Type and Dystrophic mdx Mouse Muscle Tanya R. Cully 1 , Joshua N. Edwards 1 , Robyn M. Murphy 2 , Bradley S. Launikonis 1 . 1 University of Queensland, Brisbane, Australia, 2 La Trobe University, Melbourne, Australia. Store-operated Ca 2þ entry (SOCE) is a ubiquitously expressed signalling sys- tem that is highly specialized in skeletal muscle. ‘‘Deregulated’’ SOCE had been proposed as a pathway for Ca 2þ entry into dystrophic muscle that leads to fibre degradation. We recently showed that this mechanism remains tightly regulated in mdx mouse muscle but the integral SOCE proteins, STIM1 and Orai1, are upregulated 3-fold (Edwards et al., 2010). We now report that the newly identified isoform STIM1L (Darbellay et al., 2011) is upregulated 1.8- fold in mdx muscle. We found SOCE recorded in skinned fibres was 2-fold greater in mdx compared with WT for the same SR Ca 2þ release amplitude. However cytoplasmic fluo-4 transients in depleted intact fibres showed SOCE in the absence of a sarcoplasmic reticulum (SR) Ca 2þ pump blocker to be of re- duced influx rate in mdx compared to WT fibres. A similar level of SR Ca2þ reloading was detemined in both muscle types following SOCE deactivation. Fura-2 imaging in intact fibres in the presence of 50 mM cyclopiazonic acid (CPA) and no external Ca 2þ showed that more Ca 2þ remained in the cytoplasm of mdx compared to WT fibres following SR depletion suggesting that Ca2þ extrusion by the plasma membrane Ca 2þ -ATPase (PMCA) is restricted in mdx. This helps explain reduced SOCE in intact mdx fibres, as the washout of CPA following SR depletion resulted in the greater amount of trapped cyto- plasmic Ca 2þ re-entering SR to cause a greater degree of SOCE deactivation be- fore externally applied Ca 2þ could enter the fibre. These results suggest that store-dependent Ca 2þ influx is greater and PMCA is restricted in its capacity to extrude Ca 2þ in mdx compared to WT fibres. 1848-Pos Board B618 Skeletal Respiratory Muscle in a Model of Chronic Obstructive Pulmonary Disorder Patrick Robison 1 , Erick Hernandez-Ochoa 1 , Ramzi J. Khairallah 1 , Thomas E. Sussan 2 , Christopher W. Ward 3 , Shyam Biswal 2 , Martin F. Schneider 1 . 1 University of Maryland School of Medicine, Baltimore, MD, USA, 2 Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD, USA, 3 University of Maryland School of Nursing, Baltimore, MD, USA. In Chronic Obstructive Pulmonary Disorder (COPD), mechanical deformations or work overload of the respiratory skeletal muscles may lead to adaptive or mal- adaptive muscle responses. Sarcomeric deletion, myosin type changes and met- abolic alterations have all been observed in respiratory muscles. While the diaphragm has been extensively studied, other respiratory muscles have not been closely examined in this context. Here we apply a new technique for gen- erating primary cultures of intercostal muscle (ITC) fibers to an established mouse model of COPD, thus isolating changes in individual muscle fibers from the general defects in pulmonary function known to occur in COPD. Single ITC fibers were enzymatically dissociated and either loaded with fluorescent cal- cium indicators or mounted using a novel muscle stretch tool to measure their length-tension relationship as well as passive tension over sarcomere length. Us- ing the ratiometric indicator indo-1, we found a substantial reduction in peak cal- cium of action potential induced calcium transients in COPD ITC (405/485 ratio; 1.22 vs. 0.95, p=0.003 vs control), but no significant changes in resting calcium concentration (p=0.33). Data taken with MagFluo-4 agree qualitatively with the indo-1 data and show a shortening of the overall decay phase in the ITC fibers of COPD animals. To our knowledge, these data represent the first ex-vivo assess- ment of the intercostal muscles in COPD. Supported by NIH Grants R01- AR056477, R01-AR055099, T32-AR007592 and T32-HL072751. 1849-Pos Board B619 Detection of Sub-Cellular Reactive Oxygen Species in Skeletal Muscle Poulami Basu Thakur, George G. Rodney. Baylor College of Medicine, Houston, TX, USA. Our recent studies suggest that sub-cellular site-specific ROS production is likely to play an important role in the response of skeletal muscle to oxidative stress. Using redox active probes we have targeted NADPH oxidase (Nox2), mitochondria, cytosolic glutathione, and the endoplasmic/sarcoplasmic reticu- lum to investigate these various sources of ROS under physiological and path- ophysiological conditions. We show that mechanical stretch increases ROS produced from Nox2. Repetitive contractile activity increases ROS produced from Nox2 and results in activation of the cytosolic glutathione redox cycle. Under conditions that promote ER stress and apoptosis our ER/SR redox probe becomes more oxidized. Understanding the sub-cellular signaling pathways by which oxidants influence muscle function will allow for the development of tar- geted therapeutic interventions to combat the deleterious effects of hypoxia/re- oxygenation, sustained contractile activity as well as skeletal muscle diseases. 1850-Pos Board B620 Enriching Satellite Cells with a2d1 Promotes Differentiation Tammy Tamayo, Jesu ´s Garcı ´a. UIC, Chicago, IL, USA. Cell transplants into skeletal muscle of patients with muscular dystrophy are lim- ited by donor cell attachment, migration, and survival in the host tissue. In animal models, despite HLA matching and a reduction of the host’s immune response, few donor cells are retained in the host muscle. Enriching cells for a surface marker that enhances ability of the cell to attach, migrate, and survive will result in improved cell retention. The purpose of this study was to determine whether a2d1-enriched primary satellite cells were better candidates for cell transplant than satellite cells without this surface marker. The a2d1 subunit is part of the L-type calcium channel but appears earlier than the other subunits. We isolated satellite cells from the hind limb muscles of neonatal mice and separated four subpopulations of cells based on the presence or absence of a2d1 and a marker of quiescence, CD34, by fluorescence activated cell sort (FACS). Satellite cells enriched with a2d1 survived in heat deactivated media past day 19, while there was no evidence of attachment or survival of cells without a2d1. In addition, cells that were positive for a2d1 and negative for CD34 demonstrated the most robust myogenesis out of all four subpopulations. Enhanced myogenesis of this subpopulation was determined by morphology, the pattern of expression of myogenic transcription factors, and the development of excitation-contraction coupling as demonstrated by the presence of L-type calcium currents and cal- cium transients. As the data relate to differentiation and survival, these results suggest that cells enriched with a2d1 and without CD34 will demonstrate greater cell retention and force generation after satellite cell transplant, which is a strong candidate for therapy in muscular dystrophy. Supported by MDA 1851-Pos Board B621 The Ca v b1 Subunit Regulates Gene Expression in Muscle Progenitor Cells Jackson Taylor 1 , Tan Zhang 1 , Laura Messi 1 , Jiang Qian 1 , Cristina Furdui 1 , Claudia Heren ˜u ´ 2 , Osvaldo Delbono 1 . 1 Wake Forest University School of Medicine, Winston-Salem, NC, USA, 2 National University of La Plata, La Plata, Argentina. CaVb subunits are traditionally considered constituents of CaV complexes (CaV1or2, CaVb, and CaV a2/d), where they localize at the plasma membrane and serve to regulate channel expression and gating properties. Recent publica- tions also show CaVb subunit localization in the nucleus. This phenomenon has been observed under a variety of conditions (different cell types, b subunit iso- forms, co-expressed proteins, etc). However, the mechanisms responsible for CaVb subunit nuclear shuttling, as well as a physiological role for this nuclear localization, remain major questions. Ongoing work in our laboratory has shown that muscle progenitor cells (myoblasts) express CaVb1 protein (but not CaV1 subunits) in both the cytoplasm and nucleus and that the loss of CaVb1 expression impairs proliferation in these cells. To better understand the mechanisms that link CaVb1 nuclear localization with control of prolifer- ation, we have conducted large-scale screening experiments designed to iden- tify which genes are directly regulated by CaVb1, as well as its protein binding partners. To test if CaVb1 may regulate gene expression, we conducted microarray experiments on RNA extracted from wild type, heterozygous, and CaVb1 -null mouse primary myoblasts. A number of gene transcripts were found to be differentially regulated based on the relative amount of CaVb1 ex- pression. To identify specific CaVb1 target genes, we performed chromatin immunoprecipitation -on-a-chip experiments to locate which promoter regions CaVb1 bound to across the entire mouse genome. Nuclear binding partners of CaVb1 were screened using affinity purification of CaVb1a-YFP from myo- blast nuclear fractions coupled with mass spectrometry. Finally, the importance of CaVb1 in embryonic myogenesis was explored in CaVb1 -null mice. Our results support the idea of CaVb subunits acting as transcription factors and regulating gene expression independently from CaV’s, and suggest these functions may be particularly important to progenitor cell growth. 1852-Pos Board B622 Mechanism of HMG-CoA Reductase Inhibitor-Induced Contractile Dysfunction in Rat Cultured Skeletal Muscle Kazuho Sakamoto, Syoko Tanaka, Masaya Yamamoto, Anna Mizuno, Tomoyuki Ono, Satoshi Waguri, Junko Kimura. Fukushima Medical University, Fukushima, Japan. HMG-CoA reductase inhibitors (statins), cholesterol-lowering drugs, cause contractile dysfunction of skeletal muscles as an adverse effect. We investi- gated the mechanism underlying this effect in cultured myofibers isolated Monday, February 27, 2012 365a
Transcript of Enriching Satellite Cells with α2δ1 Promotes Differentiation
Monday, February 27, 2012 365a
1847-Pos Board B617Different Capacity for Store-Operated Ca2D Entry and Ca2D ExtrusionAcross the Plasma Membrane of Wild-Type and Dystrophic mdx MouseMuscleTanya R. Cully1, Joshua N. Edwards1, Robyn M. Murphy2,Bradley S. Launikonis1.1University of Queensland, Brisbane, Australia, 2La Trobe University,Melbourne, Australia.Store-operated Ca2þ entry (SOCE) is a ubiquitously expressed signalling sys-tem that is highly specialized in skeletal muscle. ‘‘Deregulated’’ SOCE hadbeen proposed as a pathway for Ca2þ entry into dystrophic muscle that leadsto fibre degradation. We recently showed that this mechanism remains tightlyregulated in mdx mouse muscle but the integral SOCE proteins, STIM1 andOrai1, are upregulated 3-fold (Edwards et al., 2010). We now report that thenewly identified isoform STIM1L (Darbellay et al., 2011) is upregulated 1.8-fold in mdx muscle. We found SOCE recorded in skinned fibres was 2-foldgreater in mdx compared with WT for the same SR Ca2þ release amplitude.However cytoplasmic fluo-4 transients in depleted intact fibres showed SOCEin the absence of a sarcoplasmic reticulum (SR) Ca2þ pump blocker to be of re-duced influx rate in mdx compared to WT fibres. A similar level of SR Ca2þreloading was detemined in both muscle types following SOCE deactivation.Fura-2 imaging in intact fibres in the presence of 50 mM cyclopiazonic acid(CPA) and no external Ca2þ showed that more Ca2þ remained in the cytoplasmof mdx compared to WT fibres following SR depletion suggesting that Ca2þextrusion by the plasma membrane Ca2þ-ATPase (PMCA) is restricted inmdx. This helps explain reduced SOCE in intact mdx fibres, as the washoutof CPA following SR depletion resulted in the greater amount of trapped cyto-plasmic Ca2þ re-entering SR to cause a greater degree of SOCE deactivation be-fore externally applied Ca2þ could enter the fibre. These results suggest thatstore-dependent Ca2þ influx is greater and PMCA is restricted in its capacityto extrude Ca2þ in mdx compared to WT fibres.
1848-Pos Board B618Skeletal Respiratory Muscle in a Model of Chronic ObstructivePulmonary DisorderPatrick Robison1, Erick Hernandez-Ochoa1, Ramzi J. Khairallah1,Thomas E. Sussan2, Christopher W. Ward3, Shyam Biswal2,Martin F. Schneider1.1University of Maryland School of Medicine, Baltimore, MD, USA,2Johns Hopkins University Bloomberg School of Public Health, Baltimore,MD, USA, 3University of Maryland School of Nursing, Baltimore, MD, USA.In Chronic Obstructive Pulmonary Disorder (COPD), mechanical deformationsorwork overload of the respiratory skeletalmusclesmay lead to adaptive ormal-adaptive muscle responses. Sarcomeric deletion, myosin type changes and met-abolic alterations have all been observed in respiratory muscles. While thediaphragm has been extensively studied, other respiratory muscles have notbeen closely examined in this context. Here we apply a new technique for gen-erating primary cultures of intercostal muscle (ITC) fibers to an establishedmouse model of COPD, thus isolating changes in individual muscle fibersfrom the general defects in pulmonary function known to occur in COPD. SingleITCfiberswere enzymatically dissociated and either loadedwith fluorescent cal-cium indicators or mounted using a novel muscle stretch tool to measure theirlength-tension relationship as well as passive tension over sarcomere length. Us-ing the ratiometric indicator indo-1,we found a substantial reduction in peak cal-ciumof action potential induced calcium transients inCOPD ITC (405/485 ratio;1.22 vs. 0.95, p=0.003 vs control), but no significant changes in resting calciumconcentration (p=0.33). Data taken with MagFluo-4 agree qualitatively with theindo-1 data and show a shortening of the overall decay phase in the ITC fibers ofCOPD animals. To our knowledge, these data represent the first ex-vivo assess-ment of the intercostal muscles in COPD. Supported by NIH Grants R01-AR056477, R01-AR055099, T32-AR007592 and T32-HL072751.
1849-Pos Board B619Detection of Sub-Cellular Reactive Oxygen Species in Skeletal MusclePoulami Basu Thakur, George G. Rodney.Baylor College of Medicine, Houston, TX, USA.Our recent studies suggest that sub-cellular site-specific ROS production islikely to play an important role in the response of skeletal muscle to oxidativestress. Using redox active probes we have targeted NADPH oxidase (Nox2),mitochondria, cytosolic glutathione, and the endoplasmic/sarcoplasmic reticu-lum to investigate these various sources of ROS under physiological and path-ophysiological conditions. We show that mechanical stretch increases ROSproduced from Nox2. Repetitive contractile activity increases ROS producedfrom Nox2 and results in activation of the cytosolic glutathione redox cycle.Under conditions that promote ER stress and apoptosis our ER/SR redox probebecomes more oxidized. Understanding the sub-cellular signaling pathways by
which oxidants influence muscle function will allow for the development of tar-geted therapeutic interventions to combat the deleterious effects of hypoxia/re-oxygenation, sustained contractile activity as well as skeletal muscle diseases.
1850-Pos Board B620Enriching Satellite Cells with a2d1 Promotes DifferentiationTammy Tamayo, Jesus Garcıa.UIC, Chicago, IL, USA.Cell transplants into skeletalmuscle of patientswithmuscular dystrophy are lim-ited bydonor cell attachment,migration, and survival in the host tissue. In animalmodels, despite HLA matching and a reduction of the host’s immune response,few donor cells are retained in the host muscle. Enriching cells for a surfacemarker that enhances ability of the cell to attach, migrate, and survive will resultin improved cell retention. The purpose of this study was to determine whethera2d1-enriched primary satellite cells were better candidates for cell transplantthan satellite cells without this surface marker. The a2d1 subunit is part of theL-type calcium channel but appears earlier than the other subunits. We isolatedsatellite cells from the hind limb muscles of neonatal mice and separated foursubpopulations of cells based on the presence or absence of a2d1 and a markerof quiescence, CD34, by fluorescence activated cell sort (FACS). Satellite cellsenriched with a2d1 survived in heat deactivated media past day 19, while therewas no evidence of attachment or survival of cells without a2d1. In addition,cells that were positive for a2d1 and negative for CD34 demonstrated themost robust myogenesis out of all four subpopulations. Enhanced myogenesisof this subpopulation was determined by morphology, the pattern of expressionofmyogenic transcription factors, and the development of excitation-contractioncoupling as demonstrated by the presence of L-type calcium currents and cal-cium transients. As the data relate to differentiation and survival, these resultssuggest that cells enrichedwitha2d1 andwithoutCD34will demonstrate greatercell retention and force generation after satellite cell transplant, which is a strongcandidate for therapy in muscular dystrophy.Supported by MDA
1851-Pos Board B621The Cavb1 Subunit Regulates Gene Expression in Muscle Progenitor CellsJackson Taylor1, Tan Zhang1, Laura Messi1, Jiang Qian1, Cristina Furdui1,Claudia Herenu2, Osvaldo Delbono1.1Wake Forest University School of Medicine, Winston-Salem, NC, USA,2National University of La Plata, La Plata, Argentina.CaVb subunits are traditionally considered constituents of CaV complexes(CaV1or2, CaVb, and CaV a2/d), where they localize at the plasma membraneand serve to regulate channel expression and gating properties. Recent publica-tions also show CaVb subunit localization in the nucleus. This phenomenon hasbeen observed under a variety of conditions (different cell types, b subunit iso-forms, co-expressed proteins, etc). However, the mechanisms responsible forCaVb subunit nuclear shuttling, as well as a physiological role for this nuclearlocalization, remain major questions. Ongoing work in our laboratory hasshown that muscle progenitor cells (myoblasts) express CaVb1 protein (butnot CaV1 subunits) in both the cytoplasm and nucleus and that the loss ofCaVb1 expression impairs proliferation in these cells. To better understandthe mechanisms that link CaVb1 nuclear localization with control of prolifer-ation, we have conducted large-scale screening experiments designed to iden-tify which genes are directly regulated by CaVb1, as well as its protein bindingpartners. To test if CaVb1 may regulate gene expression, we conductedmicroarray experiments on RNA extracted from wild type, heterozygous, andCaVb1 -null mouse primary myoblasts. A number of gene transcripts werefound to be differentially regulated based on the relative amount of CaVb1 ex-pression. To identify specific CaVb1 target genes, we performed chromatinimmunoprecipitation -on-a-chip experiments to locate which promoter regionsCaVb1 bound to across the entire mouse genome. Nuclear binding partners ofCaVb1 were screened using affinity purification of CaVb1a-YFP from myo-blast nuclear fractions coupled with mass spectrometry. Finally, the importanceof CaVb1 in embryonic myogenesis was explored in CaVb1 -null mice. Ourresults support the idea of CaVb subunits acting as transcription factors andregulating gene expression independently from CaV’s, and suggest thesefunctions may be particularly important to progenitor cell growth.
1852-Pos Board B622Mechanism of HMG-CoA Reductase Inhibitor-Induced ContractileDysfunction in Rat Cultured Skeletal MuscleKazuho Sakamoto, Syoko Tanaka, Masaya Yamamoto, Anna Mizuno,Tomoyuki Ono, Satoshi Waguri, Junko Kimura.Fukushima Medical University, Fukushima, Japan.HMG-CoA reductase inhibitors (statins), cholesterol-lowering drugs, causecontractile dysfunction of skeletal muscles as an adverse effect. We investi-gated the mechanism underlying this effect in cultured myofibers isolated
