Mutation of δ-Sarcoglycan Is Associated with Ca2+-Dependent Vascular Remodeling in the Syrian Hamster
2007; Elsevier BV; Volume: 171; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2007.070054
ISSN1525-2191
AutoresLarissa Lipskaia, Caroline Pinet, Yves Fromes, Stéphane N. Hatem, Isabelle Cantaloube, Alain Coulombe, Anne‐Marie Lompré,
Tópico(s)Cardiac Fibrosis and Remodeling
ResumoWe examined whether mutation of the δ-sarcoglycan gene, which causes dilated cardiomyopathy, also alters the vascular smooth muscle cell (VSMC) phenotype and arterial function in the Syrian hamster CHF 147. Thoracic aorta media thickness showed marked variability in diseased hamsters with zones of atrophy and hypertrophied segments. CHF-147 VSMCs displayed a proliferating/"synthetic" phenotype characterized by the absence of the smooth muscle myosin heavy chain SM2, dystrophin, and Ca2+-handling proteins, and the presence of cyclin D1. In freshly isolated VSMCs from CHF 147 hamsters, voltage-independent basal Ca2+ channels showed enhanced activity similar to that in proliferating wild-type (WT) cells. The transcription factor NFAT (nuclear factor of activated T cells) was spontaneously active in freshly isolated CHF 147 VSMCs, as in proliferating VSMCs from WT hamsters. Mibefradil inhibited B-type channels, NFAT activity, and VSMC proliferation. CHF 147 hamsters had abundant apoptotic cells distributed in patches along the aorta, and clusters of inactive mitochondria were observed in 25% of isolated CHF 147 cells, whereas no such clusters were seen in WT cells. In conclusion, mutation of the δ-sarcoglycan gene increases plasma membrane permeability to Ca2+, activates the Ca2+-regulated transcription factor NFAT, and leads to spontaneous mitochondrial aggregation, causing abnormal VSMC proliferation and apoptosis. We examined whether mutation of the δ-sarcoglycan gene, which causes dilated cardiomyopathy, also alters the vascular smooth muscle cell (VSMC) phenotype and arterial function in the Syrian hamster CHF 147. Thoracic aorta media thickness showed marked variability in diseased hamsters with zones of atrophy and hypertrophied segments. CHF-147 VSMCs displayed a proliferating/"synthetic" phenotype characterized by the absence of the smooth muscle myosin heavy chain SM2, dystrophin, and Ca2+-handling proteins, and the presence of cyclin D1. In freshly isolated VSMCs from CHF 147 hamsters, voltage-independent basal Ca2+ channels showed enhanced activity similar to that in proliferating wild-type (WT) cells. The transcription factor NFAT (nuclear factor of activated T cells) was spontaneously active in freshly isolated CHF 147 VSMCs, as in proliferating VSMCs from WT hamsters. Mibefradil inhibited B-type channels, NFAT activity, and VSMC proliferation. CHF 147 hamsters had abundant apoptotic cells distributed in patches along the aorta, and clusters of inactive mitochondria were observed in 25% of isolated CHF 147 cells, whereas no such clusters were seen in WT cells. In conclusion, mutation of the δ-sarcoglycan gene increases plasma membrane permeability to Ca2+, activates the Ca2+-regulated transcription factor NFAT, and leads to spontaneous mitochondrial aggregation, causing abnormal VSMC proliferation and apoptosis. Disruption of the plasma membrane-associated sarcoglycan-sarcospan complex as a result of genetic defects causes muscular dystrophy and/or cardiomyopathy in humans (limb-girdle muscular dystrophy).1Tsubata S Bowles KR Vatta M Zintz C Titus J Muhonen L Bowles NE Towbin JA Mutations in the human delta-sarcoglycan gene in familial and sporadic dilated cardiomyopathy.J Clin Invest. 2000; 106: 655-662Crossref PubMed Scopus (308) Google Scholar There are six sarcoglycan family members: α-, β-, γ-, δ-, ε-, and ζ-sarcoglycan.2Wheeler MT Korcarz CE Collins KA Lapidos KA Hack AA Lyons MR Zarnegar S Earley JU Lang RM McNally EM Secondary coronary artery vasospasm promotes cardiomyopathy progression.Am J Pathol. 2004; 164: 1063-1071Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar In hamster and mouse models, δ-sarcoglycan gene deletion results in myopathy of cardiac and skeletal muscles, with focal areas of necrosis3Sakamoto A Ono K Abe M Jasmin G Eki T Murakami Y Masaki T Toyo-oka T Hanaoka F Both hypertrophic and dilated cardiomyopathies are caused by mutation of the same gene, delta-sarcoglycan, in hamster: an animal model of disrupted dystrophin-associated glycoprotein complex.Proc Natl Acad Sci USA. 1997; 94: 13873-13878Crossref PubMed Scopus (271) Google Scholar, 4Hack AA Cordier L Shoturma DI Lam MY Sweeney HL McNally EM Muscle degeneration without mechanical injury in sarcoglycan deficiency.Proc Natl Acad Sci USA. 1999; 96: 10723-10728Crossref PubMed Scopus (108) Google Scholar, 5Coral-Vazquez R Cohn RD Moore SA Hill JA Weiss RM Davisson RL Straub V Barresi R Bansal D Hrstka RF Williamson R Campbell KP Disruption of the sarcoglycan-sarcospan complex in vascular smooth muscle: a novel mechanism for cardiomyopathy and muscular dystrophy.Cell. 1999; 98: 465-474Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar and autophagic cardiomyocyte death.6Miyata S Takemura G Kawase Y Li Y Okada H Maruyama R Ushikoshi H Esaki M Kanamori H Li L Misao Y Tezuka A Toyo-Oka T Minatoguchi S Fujiwara T Fujiwara H Autophagic cardiomyocyte death in cardiomyopathic hamsters and its prevention by granulocyte colony-stimulating factor.Am J Pathol. 2006; 168: 386-397Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar Most of the studies on δ-sarcoglycan-deficient animals have been conducted on skeletal and cardiac muscles. The few studies on smooth muscle concerned the vasospasm of coronary arteries, but there are no data on the peripheral vessels. Sarcoglycans are transmembrane components of the dystrophin-glycoprotein complex, which links the cytoskeleton to the extracellular matrix.7Wheeler MT McNally EM Sarcoglycans in vascular smooth and striated muscle.Trends Cardiovasc Med. 2003; 13: 238-243Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar At the cellular level, disruption of the dystrophin-glycoprotein complex leads to increased permeability to divalent cations through channel-blocker-sensitive pathways and entry of calcium via nonspecific cation channels.8Turner PR Fong PY Denetclaw WF Steinhardt RA Increased calcium influx in dystrophic muscle.J Cell Biol. 1991; 115: 1701-1712Crossref PubMed Scopus (228) Google Scholar, 9Fong PY Turner PR Denetclaw WF Steinhardt RA Increased activity of calcium leak channels in myotubes of Duchenne human and mdx mouse origin.Science. 1990; 250: 673-676Crossref PubMed Scopus (282) Google Scholar, 10Iwata Y Katanosaka Y Arai Y Komamura K Miyatake K Shigekawa M A novel mechanism of myocyte degeneration involving the Ca2+-permeable growth factor-regulated channel.J Cell Biol. 2003; 161: 957-967Crossref PubMed Scopus (216) Google Scholar, 11Tutdibi O Brinkmeier H Rudel R Fohr KJ Increased calcium entry into dystrophin-deficient muscle fibres of MDX and ADR-MDX mice is reduced by ion channel blockers.J Physiol. 1999; 515: 859-868Crossref PubMed Scopus (112) Google Scholar The mechanisms of this enhanced Ca2+ influx are not fully understood, but changes in the activity of several Ca2+ channels have been described in dystrophin-deficient myocytes.12Vandebrouck A Ducret T Basset O Sebille S Raymond G Ruegg U Gailly P Cognard C Constantin B Regulation of store-operated calcium entries and mitochondrial uptake by minidystrophin expression in cultured myotubes.FASEB J. 2006; 20: 136-138Crossref PubMed Scopus (53) Google Scholar, 13Vandebrouck C Martin D Colson-Van Schoor M Debaix H Gailly P Involvement of TRPC in the abnormal calcium influx observed in dystrophic (mdx) mouse skeletal muscle fibers.J Cell Biol. 2002; 158: 1089-1096Crossref PubMed Scopus (278) Google Scholar, 14Johnson BD Scheuer T Catterall WA Convergent regulation of skeletal muscle Ca2+ channels by dystrophin, the actin cytoskeleton, and cAMP-dependent protein kinase.Proc Natl Acad Sci USA. 2005; 102: 4191-4196Crossref PubMed Scopus (42) Google Scholar, 15Nakamura TY Iwata Y Sampaolesi M Hanada H Saito N Artman M Coetzee WA Shigekawa M Stretch-activated cation channels in skeletal muscle myotubes from sarcoglycan-deficient hamsters.Am J Physiol. 2001; 281: C690-C699Google Scholar Dystrophin, through PDZ domain-containing adaptor proteins known as syntrophins, can link the cytoskeleton to various membrane proteins carrying a PDZ domain, including ion channels.16Vandebrouck A Sabourin J Rivet J Balghi H Sebille S Kitzis A Raymond G Cognard C Bourmeyster N Constantin B Regulation of capacitative calcium entries by alpha1-syntrophin: association of TRPC1 with dystrophin complex and the PDZ domain of alpha1-syntrophin.FASEB J. 2007; 21: 608-617Crossref PubMed Scopus (90) Google Scholar This cytoskeleton-ion channel interaction contributes to receptor/channel localization and to the regulation of voltage-, ligand-, and store-operated ion channels. Indeed, restoration of functional dystrophin-sarcoglycan complex formation by gene transfer of minidystrophin or δ-sarcoglycan normalizes ion channel function in dystrophic myocytes.10Iwata Y Katanosaka Y Arai Y Komamura K Miyatake K Shigekawa M A novel mechanism of myocyte degeneration involving the Ca2+-permeable growth factor-regulated channel.J Cell Biol. 2003; 161: 957-967Crossref PubMed Scopus (216) Google Scholar, 12Vandebrouck A Ducret T Basset O Sebille S Raymond G Ruegg U Gailly P Cognard C Constantin B Regulation of store-operated calcium entries and mitochondrial uptake by minidystrophin expression in cultured myotubes.FASEB J. 2006; 20: 136-138Crossref PubMed Scopus (53) Google Scholar, 17Marchand E Constantin B Balghi H Claudepierre MC Cantereau A Magaud C Mouzou A Raymond G Braun S Cognard C Improvement of calcium handling and changes in calcium-release properties after mini- or full-length dystrophin forced expression in cultured skeletal myotubes.Exp Cell Res. 2004; 297: 363-379Crossref PubMed Scopus (19) Google Scholar, 18Friedrich O Both M Gillis JM Chamberlain JS Fink RH Mini-dystrophin restores L-type calcium currents in skeletal muscle of transgenic mdx mice.J Physiol. 2004; 555: 251-265Crossref PubMed Scopus (49) Google ScholarIn vascular smooth muscle cells (VSMCs), Ca2+ homeostasis not only controls vessel tone but also defines the cell phenotype (from quiescent/contractile to proliferating/"synthetic"). The proliferating/synthetic phenotype is associated with a reduction in contractile performance owing to the loss of adult isoforms of contractile proteins and dystrophin.19Quignard JF Harricane MC Menard C Lory P Nargeot J Capron L Mornet D Richard S Transient down-regulation of L-type Ca(2+) channel and dystrophin expression after balloon injury in rat aortic cells.Cardiovasc Res. 2001; 49: 177-188Crossref PubMed Scopus (47) Google Scholar Moreover, proliferating VSMCs lose RyR and sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) 2a,20Vallot O Combettes L Jourdon P Inamo J Marty I Claret M Lompre AM Intracellular Ca(2+) handling in vascular smooth muscle cells is affected by proliferation.Arterioscler Thromb Vasc Biol. 2000; 20: 1225-1235Crossref PubMed Scopus (79) Google Scholar LTCC (L-type Ca2+ channels) are replaced by TTCC (T-type Ca2+ channels), and SOC (store-operated channels) as well as TRPCs (transient receptor potential protein family C) are up-regulated.21Lipskaia L Lompré AM Alteration in temporal kinetics of Ca2+ signaling and control of growth and proliferation.Biol Cell. 2004; 96: 55-68Crossref PubMed Scopus (158) Google Scholar This results in an increased cytosolic Ca2+ concentration and changes in the spatiotemporal pattern of Ca2+ signals, which can alter gene expression by activating various protein kinases and phosphatases and Ca2+-sensitive transcription factors.22Bading H Ginty DD Greenberg ME Regulation of gene expression in hippocampal neurons by distinct calcium signaling pathways.Science. 1993; 260: 181-186Crossref PubMed Scopus (952) Google Scholar, 23Dolmetsch RE Lewis RS Goodnow CC Healy JI Differential activation of transcription factors induced by Ca2+ response amplitude and duration.Nature. 1997; 386: 855-858Crossref PubMed Scopus (1548) Google Scholar For instance, a sustained increase in cytosolic Ca2+ is necessary to activate calcineurin, a Ca2+/calmodulin-dependent serine/threonine-specific protein phosphatase 2B (PP2B) that dephosphorylates nuclear factor of activated T cells (NFAT), inducing its translocation into the nucleus and transcriptional activation. NFAT is involved in the control of cell cycle-related proteins required for VSMC proliferation24Lipskaia L del Monte F Capiod T Yacoubi S Hadri L Hours M Hajjar RJ Lompre AM Sarco/endoplasmic reticulum Ca2+-ATPase gene transfer reduces vascular smooth muscle cell proliferation and neointima formation in the rat.Circ Res. 2005; 97: 488-495Crossref PubMed Scopus (81) Google Scholar, 25Liu Z Zhang C Dronadula N Li Q Rao GN Blockade of nuclear factor of activated T cells activation signaling suppresses balloon injury-induced neointima formation in a rat carotid artery model.J Biol Chem. 2005; 280: 14700-14708Crossref PubMed Scopus (83) Google ScholarThe aim of this study was to determine the consequences of δ-sarcoglycan gene mutation on vessels of CHF 147 myopathic Syrian hamsters. We postulated that alterations of the dystrophin/sarcoglycan complex would be associated with enhanced transmembrane Ca2+ influx and with activation of Ca2+-dependent processes in VSMCs.Materials and MethodsAnimalsAnimals were treated in accordance with institutional guidelines. The study was performed on thoracic aortas from 6- to 12-month-old male and female cardiomyopathic Syrian hamsters of the strain CHF 147 (raised by INSERM U582, Paris, France) and their control Golden hamsters (WT) obtained from Janvier-France breeders.MaterialsAll media, sera, and antibiotics were from Invitrogen (Cergy Pontoise, France). All chemicals were from Sigma-Aldrich (Saint Quentin Fallavier, France). The following primary antibodies were used: anti-SERCA 2a and anti-SERCA 2b (provided by Dr. F. Wuytack, University of Leuven, Leuven, Belgium),26Eggermont JA Wuytack F Verbist J Casteels R Expression of endoplasmic-reticulum Ca2(+)-pump isoforms and of phospholamban in pig smooth-muscle tissues.Biochem J. 1990; 271: 649-653Crossref PubMed Scopus (121) Google Scholar anti-RyR (provided by I. Marty, INSERM U607, Département Réponse et Dynamique Cellulaires-Grenoble, France),27Marty I Robert M Villaz M De Jongh K Lai Y Catterall WA Ronjat M Biochemical evidence for a complex involving dihydropyridine receptor and ryanodine receptor in triad junctions of skeletal muscle.Proc Natl Acad Sci USA. 1994; 91: 2270-2274Crossref PubMed Scopus (136) Google Scholar anti-SM2 (Ab 683; Abcam plc, Cambridge, UK), anti-NM-MHC-B (Ab 684; Abcam), anti-dystrophin (NCL-DYS2; Novocastra, Newcastle, UK), anti-caveolin 1 (ab2910; Abcam), anti-PMCA (ab2825; Abcam), anti-cyclin D1 (556470; BD Biosciences), and anti-NFATc1 (K-18; Santa Cruz Biotechnology, Santa Cruz, CA).Histology and Immunofluorescence StudiesMedia thickness was measured on hematoxylin and eosin-stained frozen cross sections with a computer-based morphometric system (Lucia; Nikon, Tokyo, Japan). Ten measurements were made on each section, and five discontinuous sections were analyzed in each animal. Ten CHF 147 and 10 WT hamsters were studied.Apoptosis was analyzed by terminal deoxynucleotidyl transferase dUTP nick-end labeling staining of fixed cross sections with a standard protocol (ApopTag Red; Serologicals Corporation, Norcross, GA). Immunocytochemical analysis was applied to methanol-fixed cells or acetone-fixed sections according to a standard protocol (Santa Cruz Biotechnology). Proteins were visualized by using either secondary antibodies directly conjugated to Texas Red or the biotin/streptavidin-Texas Red-conjugated amplification method (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Nuclei were labeled with Hoescht.Cell CultureVSMCs were isolated from the thoracic aorta of Syrian CHF 147 and WT hamsters and cultured as described elsewhere.28Lipskaia L Pourci ML Delomenie C Combettes L Goudouneche D Paul JL Capiod T Lompre AM Phosphatidylinositol 3-kinase and calcium-activated transcription pathways are required for VLDL-induced smooth muscle cell proliferation.Circ Res. 2003; 92: 1115-1122Crossref PubMed Scopus (60) Google Scholar Proliferation was measured by using the CellTiter96 Cell Proliferation Assay kit (Promega, Charbonnières, France).Single-Channel Recordings and Data AnalysisExperiments were performed with the cell-attached and/or inside-out patch-clamp configuration. Patch pipettes (10 to 15 mol/LΩ) were pulled from borosilicate glass capillaries (Corning Kovar Sealing 7052; WPI, Sarasota, FL). Currents were recorded with an Axopatch 200B amplifier (Axon Instruments, Foster City, CA). Channel activity (relative mean membrane patch current) was calculated as previously reported.29Coulombe A Lefevre IA Baro I Coraboeuf E Barium- and calcium-permeable channels open at negative membrane potentials in rat ventricular myocytes.J Membr Biol. 1989; 111: 57-67Crossref PubMed Scopus (44) Google Scholar All experiments were conducted at room temperature (20 to 24°C). The superfusion control and bath solutions contained 128 mmol/L potassium aspartate, 2 mmol/L KCl, 1 mmol/L BaCl2, 5 mmol/L ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 10 mmol/L HEPES, and 10 mmol/L glucose; pH was adjusted to 7.4 with KOH. The pipette solution contained 48 mmol/L BaCl2 and 10 mmol/L HEPES; pH was adjusted to 7.4 with CsOH. When the patch pipette contained 48 mmol/L Ba2+, and the patch membrane potential was continuously held at −80 mV (no voltage pulses being applied), spontaneous inward currents corresponding to channel opening were recorded. Ba2+ was used as charge carrier because it is generally considered that Ba2+ ions are more permeable than Ca2+ ions through several types of Ca2+ channels (L- and T-type), and this property seems to hold for B-type Ca2+ channels.30Lefevre T Coraboeuf E Ghazi A Coulombe A Divalent cation channels activated by phenothiazines in membrane of rat ventricular myocytes.J Membr Biol. 1995; 147: 147-158Crossref PubMed Scopus (17) Google Scholar Because Ba2+ blocks practically all known K+, Na+, and Cl− channels, it is useful for studying Ca2+ channels. We also wanted to avoid activating Ca2+-activated K+ or Cl− channels by Ca2+ flowing through these channels. We used blockade by eosin applied to the internal face of the membrane patch to confirm the presence of B-type channels.Confocal MicroscopySlides were examined with a Zeiss LSM-510 laser scanning confocal microscope (Carl Zeiss GmbH, Jena, Germany) equipped with a 30-mW argon laser and a 1-mW helium-neon laser, using a 20X/NA 0.75 Plan-Apochromat or 63X/NA 1.40 Plan Apochromat oil immersion objective. Green fluorescence was observed with a 505- to 550-nm band-pass emission filter under 488-nm laser illumination. Red fluorescence was observed with a 560-nm long-pass emission filter under 543-nm laser illumination. Pinholes were set at 1.0 Airy unit. Stacks of images were collected every 0.9 μm along the z axis. All settings were kept constant for reasons of comparability. Three median slices were projected for NFAT samples. For double immunofluorescence, dual excitation using the multitrack mode (images acquired sequentially) was obtained with the argon and He/Ne lasers.Promoter Reporter AssayCells were transfected using FuGene 6 (Roche, Basel, Switzerland) with NFAT-promoter-luciferase construct (NFAT-Luc; Stratagene, Cambridge, UK). They were cultured for 48 hours without fetal calf serum (FCS) and then treated with 10% FCS alone or together with 5 μmol/L mibefradil or 10 μmol/L cyclosporine A (CsA) for 5 hours. The luciferase activity was measured by using "the luciferase assay kit" (Promega). It was expressed as percentage of control in relative luciferase units.Mitochondrial Architecture and ActivityFor live cell confocal microscopy, cells were plated on a collagen-coated coverglass chamber (Lab-Tek II Chamber 1.5 German Cover Glass System; Nalge Nunc International, Rochester, NY) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% FCS for 24 to 48 hours. Then, the medium was replaced by Ham's F-12, with 25 mmol/L HEPES, without phenol red (Promocell, Heidelberg, Germany). To assess changes in mitochondrial membrane (Δm potential), live cells were double-labeled with the mitochondrion-sensitive Δm-independent probe MitoTracker Green (500 nmol/L, M7514; Invitrogen, Carlsbad, CA) and the Δm-sensitive dye MitoFluor Red (500 nmol/L, M22422; Invitrogen) according to manufacturers' instructions. Cells were observed in an inverted confocal microscope equipped with a chamber at 37°C. Fluorescence was recorded by means of confocal laser scanning microscopy (Leica TCS4D; Wetzlar, Germany) (λex, 490 and 598 nm; λem, 516 and 630 nm, respectively).Statistical AnalysisAll quantitative data are means ± SEM of at least three independent experiments. An unpaired t-test was used to compare means. The nonparametric Mann-Whitney test was used to compare media thickness. Differences were considered significant when P < 0.05.ResultsAltered Phenotype of CHF 147 Thoracic Aorta VSMCsIn WT hamsters, media thickness was homogenous (Figure 1, A and C), whereas in CHF 147 hamsters, there were numerous zones of atrophy and marked hypertrophy (Figure 1B). One CHF 147 animal had global hypertrophy of the media, two had aspects similar to WT, and seven were globally atrophic. The mean CHF 147 value was lower than the WT value (15.8 ± 6.9 versus 19.4 ± 2.8 μm; P < 0.001).To characterize the phenotype of CHF 147 VSMCs, aorta sections from six WT and six CHF 147 animals were labeled with antibodies specific for the contractile or synthetic phenotype and were then examined by immunofluorescence (Figure 2). The adult smooth muscle myosin heavy chain SM2 was present in aortic VSMCs from WT hamsters but was extensively replaced by the nonmuscular myosin heavy chain NM-B, a marker of the synthetic phenotype, in CHF 147 hamsters. Dystrophin, another marker of the contractile phenotype, was present in WT and absent in CHF 147 aortas, whereas caveolin-1, a specific marker of membrane caveolae, was expressed in both WT and myopathic animals.Figure 2Phenotype of smooth muscle cells from thoracic aorta. Confocal immunofluorescence. A: Expression of markers of the synthetic phenotype: nonmuscular myosin heavy chain B (NM-B) and the contractile phenotype: smooth muscle myosin heavy chain 2 (SM2) and dystrophin. Expression of caveolin 1 is also shown. B: Expression of calcium-handling proteins: SERCA 2a, SERCA 2b, RyR, and PMCA. C: Expression of cyclin D1, a marker of cell proliferation. m, media; L, lumen.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Both SERCA 2a and RyR were present in the media of WT hamsters but absent from CHF 147 animals, as previously shown in proliferating VSMCs.20Vallot O Combettes L Jourdon P Inamo J Marty I Claret M Lompre AM Intracellular Ca(2+) handling in vascular smooth muscle cells is affected by proliferation.Arterioscler Thromb Vasc Biol. 2000; 20: 1225-1235Crossref PubMed Scopus (79) Google Scholar, 28Lipskaia L Pourci ML Delomenie C Combettes L Goudouneche D Paul JL Capiod T Lompre AM Phosphatidylinositol 3-kinase and calcium-activated transcription pathways are required for VLDL-induced smooth muscle cell proliferation.Circ Res. 2003; 92: 1115-1122Crossref PubMed Scopus (60) Google Scholar The other SERCA isoform, SERCA 2b, and the plasma membrane Ca2+ pump PMCA were present in both WT and CHF 147 animals. This suggested that CHF 147 VSMCs had a synthetic phenotype. Labeling with anti-cyclin D1, a marker of proliferation, confirmed the presence of proliferating cells in the media, and especially in the luminal part of the aorta, in five out of six CHF 147 animals studied; WT aortas were negative for cyclin D1 staining (Figure 2C). Cyclin D1 expression was variable along the aorta, with zones of strong expression and other zones of no expression. Thus, the CHF aorta displayed a heterogeneous pattern of undifferentiated/proliferating VSMCs. The apparent discrepancy between the proliferative phenotype of the VSMCs and the atrophic phenotype of the vessel might be due to apoptosis in the vessel wall (see VSMCs from CHF 147 Undergo Apoptosis).Proliferative Properties of Isolated VSMCsFreshly isolated VSMCs from WT hamsters expressed markers of the differentiated phenotype, such as SM2 and SERCA 2a, whereas these markers were absent from freshly isolated VSMCs from CHF 147 hamsters while NM-B MHC was present. Freshly dissociated VSMCs from CHF 147 hamsters resembled proliferating VSMCs from WT hamsters (Figure 3). When stimulated with serum (10%), the number of VSMCs in WT increased 4.5-fold after 2 days in culture, compared with less than twofold with VSMCs from CHF 147 hamsters cultured in the same conditions (Figure 4A).Figure 3Characterization of primary VSMCs. A: Analysis of the phenotype of freshly dissociated or cultured (3 days, 10% FCS) VSMCs. Confocal immunofluorescence with specific antibodies to smooth muscle myosin heavy chain 2 (SM2), to the nonmuscle myosin heavy chain-B (NM-B), and to SERCA 2a and SERCA 2b is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Proliferation of primary VSMCs in culture. A: Analysis of the growth capacity of VSMCs from WT (▴) and CHF 147 (•) in culture. Freshly isolated cells were cultured in the presence of serum (0.1 or 10%) for 24 to 72 hours. Each point represents the mean of three to five independent experiments. At each time point the number of cells was normalized to the number of cells in 0.1% FCS (control). Values are plotted as a percentage of the control value. *P < 0.05, ***P < 0.01 WT versus CHF 147 at the same time point. B: Pharmacological analysis of the calcium signaling pathways of serum-induced proliferation. VSMCs (passages 1 to 3) were cultured for 48 hours in the presence of 0.1 or 10% FCS, either alone (control) or together with drugs at the indicated concentrations. Dil, diltiazem; Nife, nifedipine; CAI, carboxyamidotriazole; 2APB, 2-aminoethoxydiphenyl borate; Mib, mibefradil. Data are expressed as a percentage of the cell number in control wells. The results represent the average of at least four independent experiments performed in tetraplicate. **P < 0.05, ***P < 0.001 versus control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effect of Ca2+ Inhibitors on ProliferationIncreased plasma membrane permeability to Ca2+ has been described in cardiac and skeletal myocytes lacking δ-sarcoglycan.8Turner PR Fong PY Denetclaw WF Steinhardt RA Increased calcium influx in dystrophic muscle.J Cell Biol. 1991; 115: 1701-1712Crossref PubMed Scopus (228) Google Scholar, 9Fong PY Turner PR Denetclaw WF Steinhardt RA Increased activity of calcium leak channels in myotubes of Duchenne human and mdx mouse origin.Science. 1990; 250: 673-676Crossref PubMed Scopus (282) Google Scholar, 10Iwata Y Katanosaka Y Arai Y Komamura K Miyatake K Shigekawa M A novel mechanism of myocyte degeneration involving the Ca2+-permeable growth factor-regulated channel.J Cell Biol. 2003; 161: 957-967Crossref PubMed Scopus (216) Google Scholar, 11Tutdibi O Brinkmeier H Rudel R Fohr KJ Increased calcium entry into dystrophin-deficient muscle fibres of MDX and ADR-MDX mice is reduced by ion channel blockers.J Physiol. 1999; 515: 859-868Crossref PubMed Scopus (112) Google Scholar In VSMCs, elevated cytosolic Ca2+ levels induce proliferation and phenotypic changes.21Lipskaia L Lompré AM Alteration in temporal kinetics of Ca2+ signaling and control of growth and proliferation.Biol Cell. 2004; 96: 55-68Crossref PubMed Scopus (158) Google Scholar We therefore tried to detect enhanced Ca2+ entry in CHF 147 VSMCs by using various Ca2+ inhibitors (Figure 4B). Diltiazem, an inhibitor of LTCC, had no effect on the proliferation of VSMCs from WT hamsters. Nifedipine, another LTCC inhibitor, prevented WT VSMC proliferation but only at a high concentration. Mibefradil, a putative T-type Ca2+ channel blocker, completely blocked VSMC proliferation when used at a low concentration (5 μmol/L). Carboxyamidotriazole and 2-aminoethoxydiphenyl borate, two nonspecific inhibitors of capacitative calcium entry, inhibited proliferation only partially and only at high concentrations. Because mibefradil most efficiently inhibited the proliferation of WT hamster VSMCs, we also tested its effect on serum-induced proliferation of CHF 147 VSMCs. As in WT, 5 μmol/L mibefradil completely inhibited the increase in cell numbers induced by serum. Because of the small amount of cells obtained from mutant hamsters, we were unable to test the other inhibitors. These results indicate that Ca2+ entry is involved in VSMC proliferation. The calcineurin inhibitor CsA partially blocked the proliferation of VSMCs from WT hamsters (Figure 4B), suggesting the involvement of the calcium-dependent calcineurin/NFAT-signaling pathway in the proliferation of hamster VSMCs.Analysis of B-Type Channels in VSMCs from Control and CHF 147 Hamsters: Effect of MibefradilTo examine further the possibility of enhanced Ca2+ entry in CHF 147 VSMCs, we performed cell-attached and inside-out patch recordings using membranes of VSMCs freshly isolated from WT (Figure 5A) and CHF 147 hamsters (Figure 5B). In WT cells, single-channel activity (observed in 15% of membrane patches tested) exhibited rare bursts of intense activity followed by long-lasting quies
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