Feb. 2021Vol. 42 No. 12021年 2月 第 42 卷$ 第 1 期首都医科大学学报Journal of Capital Medical Universito[doi ： 10. 3969/j. U sp . 1006-7795. 2021. 01. 017]・ 临床研究 ・冠状动脉斑块旋磨联合药物洗脱支架术对不同年龄段老年钙化 治疗效果的对比分析胡 宾周玉杰！贾德安秦政许晓啥杨士伟周志明(首都医科大学附属北京安贞医院心内科北京市心肺血管疾病研究所冠心病精准治疗北京市重点实验室首都医科大学冠心病 临床诊疗与研究中心，北京100029)$摘要】目的探讨冠状动脉粥样硬化性心脏病(以下简称冠心病)严重钙化患者的年龄对冠状动脉旋磨联合药物洗脱支架治 疗效果及其安全性的影响。
方法 选取2016年1月至2017年12月连续在首都医科大学附属北京安贞医院接受冠状动脉内旋 磨和药物洗脱支架植入治疗的198例冠心病患者为研究对象，分为#65岁组116例和<65岁组82例，比较两组旋磨治疗的基线特征、并发症、1年主要不良心血管事件，包括因心肌梗死、再次血运重建及死亡。
结果 #65岁组和<65岁组围术期合并症的发生率分别为18. 9%'13. 3%,差异无统计学意义(P =0.202)。
两组患者1年主要不良心血管事件比较，差异无统计学意义 (P =0. 320) $结论 冠状动脉内旋磨和药物洗脱支架治疗老年冠心病严重钙化的效果及安全性，不受患者的年龄影响。
$关键词】旋磨术;药物洗脱支架;冠状动脉粥样硬化性心脏病$中图分类号】R541. 14Observation on effect of rotational atherectomy follower by drrg-eluting stents nmplantatnonnnpatnentsage #65 yearswnthseverecalcnfne!coronary lesnonsHu Bin ，Zhou Yujie **，Jia Dean ，Qin Zheng ，Xu Xiaohan ，Yang ShiweRZhou Zhimin 基金项目：国家重点研发计划(2017YFC0908800)，北京市医院管理局“登峰”计划专项经费资助(DFL20150601)，北京市医院管理局“使命”计划 专项经费资助(SML20180601)，北京市卫生健康委员会科技创新中心建设项目(PXM2019_026272_000006，PXM2019_026272_000005 )。
ophores populations that occur during changes in metabolism.To evaluate the impact of these changes,we have recently employed two-photon excited NADH Fluorescence Lifetime Imaging.Treat-ment with both metabolic uncouplers and inhibitors caused system-atic shifts in both the lifetime and the free to bound ratio of NADH. Assessment of metabolic state by the intensity-based and lifetime-based techniques will be compared for both monolayer cultures and the hair cells of the excised organ of Corti.Supported by NIH DC02053,NSF-EPSCoR EPS-0346476(CFD 47.076)and NIH P20RR016469from the INBRE Program of the National Center for Research Resources.Store-operated Ca++Channels2933-Pos Store-Operated Ca2+-Induced Ca2+Release Amplifies Cytosolic Ca2+ Signaling and Prevents Store Refilling in Jurkat T CellsSepehr Dadsetan1,Liudmila Zakharova1,Tadeusz F. Molinski2,Alla F.Fomina11University of California Davis,Davis,CA,USA,2University of California San Diego,San Diego,CA,USA.Board B236The precise control of many T cell functions relies on cytosolic Ca2+ dynamics which is shaped by the release of Ca2+from intracellular store and extracellular Ca2+influx via plasmalemmal store-operated Ca2+(SOC)channels.It is presumed that Ca2+influx via SOC channels is required for store refilling.However,using Fura2Ca2+ probe to simultaneously assess the store content and Ca2+dynamics in the cytosol,we demonstrated that in T lymphocytes the store refilling was inhibited in the presence of extracellular Ca2+and SOC channels activated by store depletion with cyclopiazonic acid (CPA),a reversible blocker of the sarco-endoplasmic reticulum Ca2+-ATPase.Pretreating cells with xestospongin C(10m M)or ryanodine(400m M),the antagonists of inositol1,4,5-trisphosphate (IP3R)or ryanodine(RyR)receptors,respectively,facilitated store refilling while reducing cytosolic Ca2+transients associated with SOC channel activation.These data indicate that Ca2+-induced Ca2+ release(CICR)is an essential source of Ca2+for elevation of cytosolic Ca2+concentration([Ca2+]i)upon stimulation.Consis-tently,inhibition of IP3R or RyR significantly inhibited T cell proliferation and IL2production.Finally,we observed that store refilling persisted when store-operated Ca2+entry(SOCE)was completely inhibited by1m M extracellular La3+(IC50~30nM), confirming that store can be refilled in the absence of SOCE and global[Ca2+]i elevation.Store refilling was inhibited by higher[La3 +](IC50~5m M)suggesting the existence of a store-independent store replenishing pathway that may sustain CICR.We conclude that CICR is essential mechanism regulating[Ca2+]I dynamics in T cells and that IP3R and/or RyR may represent novel pharmacologi-cal targets for manipulation of Ca2+-dependent functions of T lymphocytes.Research described in this abstract was supported by Philip Morris USA Inc.and Philip Morris International.2934-PosBoard B237WITHDRAWN2935-Pos Different STIM to Orai1 ignaling pathways between C.elegans and humanPingyong Xu,Shangbang Gao,Liangyi Chen,Jingze Lu,Tao XuInstitute of Biophysics,Beijing,China.Board B238Endoplasmic reticulum(ER)Ca2+depletion evokes Ca2+entry through Ca2+release activated Ca2+(CRAC)channel in the plasma membrane(PM).Recent studies have identified STIM1and Orai1as essential components of CRAC channel.The molecular mechanism by which STIM1is translocated and targeted on the plasma mem-brane(PM)and convey signals from the ER lumen to PM is not yet understood.Here we report that in HEK293cells the nematode Caenorhabditis elegans STIM1(C.STIM1)are pre-oligomerized in puncta at the cell periphery before store depletion,and this oligo-mization of STIM1is not sufficient for the aggregation of Orai1on the opposing PM and CRAC activation.Therefore,C.STIM1does not have the store-depletion induced translocation steps of human STIM1(H.STIM1).In contrast,the C.STIM1puncta and C.elegans Orai1functioned as a pair that can locally respond to ER store depletion and lead to activation of CRAC channel.By switching the N-and C-termini of C.STIM1to H.STIM1,we prove that the STIM1C-terminus decides its resting localization in different organisms.A mutant H.STIM1lacking the C terminal proline-rich domain and the polybasic sequence motif(PPK)that are absent in C. STIM1shows the same distribution as C.STIM1and colocalized with C.STIM1near the PM before store depletion,proving that targeting STIM1on the PM is dispensable of PPK domain.Taken together,our results suggest that:1)C.elegans uses a different signaling pathway of STIM1compared with human,but uses conserved targeting machinery in the C-terminus to trap STIM1 to the cell periphery.2)The mammalian C-terminal PPK domain evolves as an autoinhibitory mechanism by which STIM1confor-mation changes after Ca2+store depletion and exposes the PM-targeting motif for its targeting on the PM.2936-Pos Functional Comparison Of ORAI Homologs In Regulating Mammalian CRAC Channel Activity Shenyuan L.Zhang,J.Ashot Kozak,Weihua Jiang,Jing Chen,Ying Yu,Aubin Penna,Shen Wei,Victor Chi,Michael D.CahalanUCI,Irvine,CA,USA.980Meeting-AbstractBoard B239RNAi screening has identified a requirement for Stim and Orai and their mammalian homologs to produce store-operated Ca2+entry (SOCE)and CRAC channel activity.Here,we evaluate the three human homologs of Orai expressed together with STIM1separately and in combinations in HEK cells.Promiscuous heteromultimer-ization within Orai members was found by co-immunoprecipitation. Coexpressed with STIM1,Orai1induced a large inwardly rectifying Ca2+current with a form of Ca2+-induced slow inactivation.A point mutation of Orai1(E106D)drastically altered the ion selectivity of the induced CRAC current and eliminated slow inactivation while retaining an inwardly rectifying I–V ing the amplified current model of co-expressed Orai1+STIM1,we show that CRAC current can be recorded in cell-attached and inside-out patches.A C-terminal portion of Stim lacking the putative Ca2+-binding motif and the transmembrane segment interacted with Orai by co-immunoprecipitation and effectively activated Ca2+influx in S2cells in the absence of store depletion.Expression of the corresponding STIM1C-terminus,coexpressed with Orai1,was sufficient to generate CRAC current without store depletion.Fur-thermore,50m M2-APB is able to sensitize/activate exogenous Orai3channel activity without store depletion.The structural determinants responsible for the different Orai channel activities are being revealed by analyzing a series of Orai chimeras and point mutants.2937-Pos Differential Pharmacology of 2-aminoethoxydiphenyl borate(2-APB) on CRAC ChannelsAnnette J.Lis,Christine Peinelt,Andreas Beck,Suhel Parvez,Mahealani K.Monteilh-Zoller,Andrea Fleig, Reinhold PennerQueen’s Medical Center,Honolulu,HI,USA.Board B240STIM1in the endoplasmic reticulum(ER)and CRACM1(or Orai1) in the plasma membrane are essential molecular components for controlling store-operated Ca2+entry(SOCE)via CRAC channels. Combined over-expression of both proteins reconstitutes amplified CRAC currents and all three mammalian CRAC channel homologs (CRACM1,CRACM2and CRACM3)represent functional store-operated channels with distinctive properties in terms of kinetics of activation,selectivity for Ca2+,Ba2+,and different feedback regulation by intracellular Ca2+.The dissection of the various molecular components of SOCE is complicated and only few pharmacological tools are available to address this problem.Al-though2-aminoethoxydiphenyl borate(2-APB)cannot be consid-ered a SOCE-specific compound,it has previously been found to affect native CRAC channels by potentiating CRAC currents at low concentrations(<5m M)and inhibiting them at high concentrations (>10m M).We have previously reported that50m M2-APB completely blocks CRACM1,reduces CRACM2by~50%and fails to suppress CRACM3,but enhances it~7-8fold.We now have qualitatively and quantitatively assessed the effects of2-APB on both store-operated and store-independent activation of CRAC channels and reveal that the three homologs exhibit strikingly different pharmacological responses to different concentrations of 2-APB that are mediated by differential effects on the CRAC channel itself as well as through its interaction with STIM1. 2938-Pos ADAR-mediated RNA Editing of Orai1is A Key to Different Selectivity of Store-operated ChannelsVladislav V.Zarayskiy,Bo Yang,Krisztina Peter,Alan G. Herbert,Victoria M.BolotinaBoston University,Boston,MA,USA.Board B241Store-operated Ca2+entry(SOCE)is known to be mediated by store operated channels(SOCs)that may have different Ca2+selectivity. While Orai1is thought to encode Ca2+-selective SOC(CRAC)in nonexcitable cells,the molecular nature and the reasons for a poor cation selectivity of SOCs in other cell types remain a mystery.Here we presentfirst evidence that Orai1may encode different types of SOC,and the variations in their selectivity may result from post-translational modification of Orai1,with ADAR-mediated RNA editing being a key to this process.Patch-clamp,Ca2+imaging and molecular approaches were used to study Ca2+-selective CRAC in RBL-2H3cells and cation-selective SOC(cat-SOC)in SMC.We discovered that:1)siRNA knock down of Orai1impairs SOCE and whole-cell SOC currents in both,RBL and SMC cells;2)ADAR1 expression in RBL cells is significantly lower than in SMC;3) overexpression of functional ADAR1in RBL results in transforma-tion of inwardly rectifying I CRAC into a linear current,and appear-ance of significant Mn2+influx through SOCE pathway;4)siRNA knock down of endogenous ADAR1in SMC results in disappear-ance of significant Mn2+influx,and appearance of inwardly recti-fying I CRAC component of the whole-cell current.Thus,expression and functional activity of ADAR1may change the selectivity of SOC channels.We propose that while the original(un-edited)Orai1 molecule forms Ca2+-SOC(CRAC),ADAR-mediated RNA editing of specific residues may create post-transcriptional modified var-iants of Orai1that form cat-SOCs with different cation selectivity. These importantfindings resolve the long lasting controversy about the relationship between SOC channels with different cation selec-tivity,and demonstrate how one gene(Orai1)may encode different SOCs,with ADAR-dependent RNA editing adjusting their selec-tivity to the specific needs of different cell types.Membrane Receptors&Signal Transduction-I 2939-Pos Endothelial Cells Increase Tumor Cell InvasionClaudia T.Mierke1,Jose-Luis Alonso2,Jan Brabek3, Wolfgang H.Goldmann1,Ben Fabry11Universit€a t Erlangen,Erlangen,Germany,2Mass.General Hospital,Renal Unit,Charlestown,MA,USA,3Physilogy and Developmental Biology,Charles University Prague,Pra-gue,Czech Republic.Meeting-Abstract981。
ISSN: 1524-4571Copyright © 2006 American Heart Association. All rights reserved. Print ISSN: 0009-7330. OnlineTX 72514Circulation Research is published by the American Heart Association. 7272 Greenville Avenue, Dallas,DOI: 10.1161/01.RES.0000234807.16034.fe2006;99;323-331; originally published online Jun 29, 2006;Circ. Res.David Hassel, Charles Geoffrey Burns, Hugo A. Katus and Mark C. FishmanWolfgang Rottbauer, Georgia Wessels, Tillman Dahme, Steffen Just, Nicole Trano,Thick-Myofilament Assembly and Contractility of the Heart Cardiac Myosin Light Chain-2: A Novel Essential Component of/cgi/content/full/99/3/323located on the World Wide Web at:The online version of this article, along with updated information and services, is/reprints Reprints: Information about reprints can be found online atjournalpermissions@ 410-528-8550. E-mail:Fax:Kluwer Health, 351 West Camden Street, Baltimore, MD 21202-2436. Phone: 410-528-4050. Permissions: Permissions & Rights Desk, Lippincott Williams & Wilkins, a division of Wolters/subscriptions/Subscriptions: Information about subscribing to Circulation Research is online atCardiac Myosin Light Chain-2A Novel Essential Component of Thick-Myofilament Assembly andContractility of the HeartWolfgang Rottbauer,Georgia Wessels,Tillman Dahme,Steffen Just,Nicole Trano,David Hassel, Charles Geoffrey Burns,Hugo A.Katus,Mark C.FishmanAbstract—Although it is well known that mutations in the cardiac regulatory myosin light chain-2(mlc-2)gene cause hypertrophic cardiomyopathy,the precise in vivo structural and functional roles of MLC-2in the heart are only poorly understood.We have isolated a mutation in zebrafish,tell tale heart(tel m225),which selectively perturbs contractility of the embryonic heart.By positional cloning,we identified tel to encode the zebrafish mlc-2gene.In contrast to mammals, zebrafish have only1cardiac-specific mlc-2gene,which we find to be expressed in atrial and ventricular cardiomyocytes during early embryonic development,but also in the adult heart.Accordingly,loss of zMLC-2function cannot be compensated for by upregulation of another mlc-2gene.Surprisingly,ultrastructural analysis of tel cardiomyocytes reveals complete absence of organized thick myofilaments.Thus,our findings provide the first in vivo evidence that cardiac MLC-2is required for thick-filament stabilization and contractility in the vertebrate heart.(Circ Res.2006;99:323-331.)Key Words:cardiac myosin light chain-2ⅢmyofibrillogenesisⅢzebrafish heartI nherited heart muscle diseases(cardiomyopathies)in hu-mans,the leading cause of premature death in the young, are mainly caused by mutations in various sarcomeric pro-teins.Ultrastructural analysis of affected cardiomyocytes implicates disturbed myofibrillogenesis to be partially re-sponsible for altered heart function because severe disarray of myofibrils can be observed.1One of the proteins found to induce human cardiomyopathies,when mutated,is the thick-filament component cardiac regulatory myosin light chain-2 (MLC-2).2However,the precise in vivo structural and func-tional roles of MLCs in the vertebrate heart muscle are only poorly understood.Each thick filament in muscle includes2myosin heavy chains and4MLCs,which are wrapped like dumbbells around the␣-helical neck region of myosin heavy chains.3 The mlc gene families differ in size among species but may generally be characterized as regulatory(ie,phosphorylat-able)or essential.In vitro,regulatory MLCs appear to play an important part in the conversion of chemical energy into movement,specifically enhancing actin-filament sliding ve-locity.4In vivo,MLCs confer different functional roles on different muscle types.In smooth muscle,phosphorylation of regulatory MLC by myosin light chain kinase is the switch for turning on the actin-activated myosin ATPase and,hence, contraction.By contrast,in vertebrate striated muscle,the actin–myosin interaction is mainly regulated through the troponin–tropomyosin complex,and regulatory MLCs are thought to have a modulatory effect on rate and magnitude of force generation.5In the adult heart muscle,phosphorylation of regulatory MLCs increases tension production while de-creasing stretch activation response of cardiomyocytes.3,6 Two major regulatory MLC isoforms are coexpressed in the early stages of murine cardiogenesis.Disruption of MLC-2v causes dilated cardiomyopathy.7However,there is significant compensatory upregulation of atrial-specific MLC-2(MLC-2a)in the ventricular cardiomyocytes of MLC-2v–depleted mice.MLC-2a might partially compensate for the loss of MLC-2v,thereby masking an essential role of MLC-2v in vertebrate heart function.We sought here the molecular cause of an ethylnitrosourea (ENU)-induced recessive embryonic lethal zebrafish muta-tion,tell tale heart(tel m225),which selectively perturbs early embryonic heart contractility.By positional cloning,we identify tel to encode a zebrafish cardiac regulatory MLC gene(zmlc2).zmlc2is expressed throughout the heart,unlike the chamber-specific pattern noted for regulatory MLCs in mouse and human.In fact,zmlc2appears to be the only regulatory mlc gene expressed in the heart of the zebrafish. Loss of this unique zmlc completely abolishes myofibrillar assembly in a cell-autonomous fashion in tel mutant hearts,Original received March2,2006;revision received May31,2006;accepted June15,2006.From the Department of Medicine III(W.R.,G.W.,T.D.,S.J.,N.T.,D.H.,H.A.K.),University of Heidelberg,Germany;Cardiovascular Research Center(W.R.,C.G.B.,M.C.F.),Massachusetts General Hospital,and Department of Medicine,Harvard Medical School,Boston,Mass;and Novartis Institutes for BioMedical Research(M.C.F.),Cambridge,Mass.Correspondence to W.Rottbauer,Department of Medicine III,University of Heidelberg,INF410,D-69120Heidelberg,Germany.E-mail wolfgang.rottbauer@med.uni-heidelberg.de©2006American Heart Association,Inc.Circulation Research is available at DOI:10.1161/01.RES.0000234807.16034.fedemonstrating that MLC-2is essential for cardiac myofibril-logenesis in vivo.Materials and MethodsZebrafish Strains and Cell TransplantationCare and breeding of zebrafish,Danio rerio,were as described.8Cell transplantation was performed essentially as described9by inter-crossing tel m225Ϫ/ϩfish,thus yielding a wild-type to mutant embryo ratio of3:1.Genetic Mapping,Positional Cloning,and Mutation DetectionDNA from24tel m225mutant and24wild-type embryos was pooled for bulked-segregation analysis.10One hundred twenty microsatellite markers11were tested for possible linkage.The critical genomic interval for tel was defined by genotyping2249mutant tel m225 embryos for microsatellite markers in the area.Mapping information for expressed-sequence tag(EST)clones was derived from radiation hybrid maps(http://wwwmap.tuebingen.mpg.de and http://mgchd1. :8000/zfrh/beta.cgi).Recombinant fine mapping anal-ysis was performed with single-stranded conformation polymor-phisms(SSCP)from the sequence of ESTs or sequenced bacterial artificial chromosome(BAC)clones.A genomic DNA“shotgun”library of BAC159f11,which covers the tel interval,was made essentially as described.9Sequence tracings were read and processed by the Phred program.Six interconnected sequence contigs were assembled using Phrap and edited by Consed().The cDNA sequence for zmlc-2was determined by RT-PCR and rapid amplification of cDNA ends(Clontech)(GenBank accession no.AY057074).RNA from mutant and wild-type whole embryos was isolated using TRIzol Reagent(Life Technologies). Histology,Transmission Electron Microscopy, RNA Antisense In Situ Hybridization, Immunostaining,and ImmunoblottingHistological analysis and electron micrographs of fish embryos were performed as described.9Whole-mount RNA antisense in situ hybridization was performed as described9using a full-length digoxigenin-labeled zmlc-2 riboprobe.For whole-mount immunostaining,embryos were fixed in4% paraformaldehyde or Dent’s fixative.12Monoclonal antibodies against myosin heavy chain,MF20,S46,13and␣-tropomyosin (Sigma)were used.In vitro translation of wild-type and mutant MLC-2was performed using the TNT coupled reticulocyte lysate system(Promega).For immunoblotting,in vitro–translated proteins were separated by15% SDS-PAGE and transferred to polyvinylidene fluoride(PVDF) membrane.Blots were probed with anti–FLAG M2(Sigma)as primary antibody and signals detected by chemiluminescence. Injection ProceduresMorpholino-modified antisense oligonucleotide was directed against the translational start site of zebrafish cardiac mlc-2(MO-mlc-2; 5Ј-GCTTTTTTACTAGCCATGTTCACTG-3Ј)(GENETOOLS LLC).Embryos at the1-cell stage were injected with10ng of MO-mlc-2or a standard control oligonucleotide(MO-control)in0.2 mol/L KCl and0.1%phenol red.Capped mRNA was synthesized using the mMESSAGE mMASCHINE system(Ambion).Twenty-five picograms of mRNA were microinjected into1-cell-stage embryos.DNA from the BAC clone B159f11was isolated using Qiagen columns,as instructed by the manufacturer,followed by additional phenol/chloroform extraction and ethanol precipitation.BAC DNA was diluted in Danieau’s medium/0.1%phenol red,and0.5ng was microinjected into1-cell-stage embryos.Resultstel Is Required for Early Embryonic Heart Contractility in a Cell-Autonomous Fashiontel m225is an ENU-induced,fully penetrant embryonic lethal recessive mutation in zebrafish14that selectively perturbs cardiac contractility during early embryonic development. Blood circulation is never established in homozygous tel mutant embryos.Aside from pericardial edema,embryos are not noticeably affected by the lack of normal blood flow during the first week of development(Figure1A and1B). tel mutants form an apparently morphologically normal heart,in an anatomically correct position,with an endocardial and myocardial layer.By36hours post-fertilization(hpf),the 2heart chambers are distinct(atrium and ventricle),and appropriate growth of the ventricular myocardium can be observed in tel mutant embryos by72hpf(Figure1C and 1D).However,vigorous peristaltic contraction waves travers-ing the heart tube,as usually seen in wild-type embryos,are absent.By contrast,only weak,uncoordinated myocardial contractions of single heart cells can be observed in tel mutant embryos at24hpf.By48hpf,atrial and ventricular cardiomyocytes are silent and the fractional shortening of both chambers declines to0(Figure1E and1F).Interestingly, some cardiomyocytes of the rather extended and pronounced atrioventricular(AV)canal region continue to contract rhyth-mically(Movie1in the online data supplement,available at ).To examine the cell autonomy of the cardiac contraction defect in tel mutant embryos,we performed cell-transplantation experiments to generate mosaic hearts com-posed of mutant and wild-type cardiomyocytes.We found that when wild-type cells contribute to mutant hearts,they contract spontaneously and vigorously in tel ventricles and atria.In contrast,tel mutant cardiomyocytes do not contract in a wild-type host.In fact,they are thin,mechanically unstable,and bulge dyskinetically outward with each systolic contraction of the wild-type heart,forming aneurysm-like structures in the atrium and the ventricular chamber(Figure 1G and1H),similar to titin-deficient cardiomyocytes of zebrafish pickwick mutants.15Thus,the tel gene controls cardiomyocyte contractility in a cell-autonomous manner. The tel Locus Encodes a Zebrafish Cardiac Regulatory Myosin Light Chain GeneWe identified the mutation causing the recessive tel mutant phenotype by a positional walk(Figure2A).The tel mutation maps to zebrafish linkage group8between microsatellite markers Z25243and Z21115.A genetic marker derived from EST fb57f10(GenBank accession no.AI477585),linked to the tel interval by radiation hybrid(RH)mapping,16displays 1recombination event in4498tel mutant meiotic events.A BAC clone(B159f11)containing fb57f10was isolated and a “shot-gun”library constructed and sequenced.B159f11con-tains4open-reading frames.Further testing of recombination events in tel mutant embryos restricts the tel interval to the zebrafish cardiac regulatory light chain gene(zmlc-2)(Figure 2A).As in other species,zmlc-2is encoded by7exons. Conceptual translation of genomic DNA and cDNA se-324Circulation Research August4,2006quences (GenBank accession no.AY057074)reveals a high degree of homology (78%)to the human and mouse regula-tory light chain 2a (MLC-2a)protein (Figure 2B).A Splice Donor-Site Mutation Leads to Early Truncation of Zebrafish MLC-2in telTo identify the site of the mutation,the entire zebrafish coding sequence of zmlc-2from wild-type and tel mutant embryos was sequenced.Three different cDNA splice prod-ucts were detected in tel mutant cDNA,all transcripts leading to premature termination of translation before exon 3or 4,respectively.As indicated in Figure 2D,tel cDNA 1contains intron 3sequence.tel cDNA 2displays a new exon boundary exon 2/exon 4attributable to skipping of exon 3,and tel cDNA 3reveals skipping of exon 3through exon 5of the zebrafish mlc-2gene (Figure 2D),all transcripts leading to a reading-frame shift and premature stop of protein translation.To further elucidate the cause of aberrant splicing in tel mutant embryos,genomic intronic sequence was determined around exon 3.A splice donor-site mutation (g 3a)at the highly conserved position 1of the splice donor site of intron 3was detected,leading to the different splicing events in tel mutants (Figure 2C and 2D).To confirm that loss of MLC-2is indeed responsible for the tel phenotype,morpholino-modified antisense oligonucle-otides targeted against the translational start site of zebrafish cardiac mlc-2(MO-mlc-2)were injected into wild-type em-bryos (n ϭ200)at the 1-cell stage.Ninety-seven percent of the injected embryos (194/200)displayed the tel mutant pheno-type with a silent ventricle and atrium and only contractions of cardiomyocytes of the AV canal (Figure 3A through 3C and supplemental Movie 2).To test whether wild-type DNA or RNA encoding zMLC-2could suppress the tel mutant phenotype,tel mutant embryos (tel m225Ϫ/Ϫ)were injected at the 1-cell stage.After injection of 0.5ng of BAC 159f11DNA,25%of genotypically tel mutant embryos (7/28)revealed vigorous contractions of areas of the ventricular wall by 72hpf (Figure 3D).In 2rescued embryos,weak circulation of blood cells could be observed.However,none of the rescued embryos survived beyond day 8of development.Injection of capped zmlc-2mRNA also par-tially rescued up to 17%of tel mutant embryos,depending on the dose injected (Figure 3D),whereas injection of tel mutant zmlc2mRNA (tel -cDNA-1)had no effect on the tel mutant phenotype.Truncated zMLC-2Protein Does Not Accumulate in tel Mutant HeartsEven though the tel mutation interferes with only RNA processing,antisense-mediated knockdown of zmlc-2com-Figure 1.Effects of the tel m225mutation on embryonic heart function.A and B,tel mutants develop pericardial edema caused by the absence of cardiac con-tractility,whereas the development of other organ systems proceeds teral view of wild-type (wt)and tel mutant embryos at 72hpf.C and D,tel mutants display normal heart morphol-ogy.Endocardial and myocardial layers of the ventricular and atrial chamber are unaltered in tel mutants at 72hpf.Trans-verse sections of a wild-type (C)and tel mutant (D)heart region stained withhematoxylin/eosin.A indicates atrium;V,ventricle.E and F,Fractional shortening (FS)of the atrial (F )and ventricular (f )chamber of wild-type and tel mutants.Ventricular and atrial contractility is severely reduced in tel mutant hearts and decreases to 0by 60hpf.G and H,tel acts cell autonomously in cardiac myocytes.Clusters of transplanted tel cells (arrow)build aneurysm like struc-tures in a wild-type atrium (G)and ventri-cle (H)and bulge outward during atrial and ventricular systole.Rottbauer et al Cardiac Myosin Light Chain-2in Zebrafish Heart 325pletely phenocopies tel ,implicating that even in tel mutant hearts,zMLC-2function is completely abolished.Therefore,to evaluate whether the tel mutation interferes with RNA stability,we analyzed the abundance of mlc-2mRNA by whole-mount in situ antisense hybridization.As shown in Figure 4,mlc-2mRNA is severely reduced in the hearts of tel embryos at 72hpf (Figure 4A and 4B),indicating degradation of tel mutant mis-spliced zmlc-2mRNA most likely by nonsense-mediated RNA decay.17However,traces of mutant RNA are still detectable in tel hearts.To further test whether predicted truncated MLC-2protein is stable and thereby might act as a dominant negative,we generated expression plasmids of either N-or C-terminal FLAG-tagged wild-type or mutant MLC-2con-structs.However,whereas in vitro–translated N-and C-terminally tagged wild-type MLC-2accumulates in reticu-locyte lysates,neither N-nor C-terminally tagged truncated MLC-2proteins accumulated to detectable levels (Figure 4C).Because the majority of tel mutant RNA is degraded,and mutant MLC-2protein per se is not stable,lack of MLC-2rather than a dominant-negative effect of truncated MLC-2appears to cause the tel mutant heart phenotype.tel Is Not Expressed in a Cardiac Chamber–Restricted Patterntel appears to be most similar to the atrial isoform (MLC-2a)of human regulatory MLCs (Figure 2B).Human and mouse MLC-2a are known to be preferentially expressed in atrial cardiomyocytes.18However,in contrast to mammals,zmlc-2RNA is found to be expressed in both heart chambers from early embryonic development throughout adulthood (Figure 4D through 4F).Similarly,in our transgeniczebrafishFigure 2.tel encodes cardiac MLC-2and is highly conserved between zebrafish and human.A,Integrated genetic and physical map of the zebrafish tel region.The tel mutation interval is flanked by the microsatellite markers z21115and z25210.A bacterial artificial chro-mosome (BAC)clone B159f11was isolated covering the tel interval and subjected to shotgun sequencing.Sequence analysis of the tel mutation interval revealed 4zebrafish genes,1of them highly homologous to cardiac regulatory MLC.The genomic structure of zmlc-2is displayed at the bottom and the location of the tel splice donor-site mutation at the exon 2–intron 3boundary indicated by an arrow.B,Amino acid sequence alignment of zebrafish (zMLC-2)with mouse atrial MLC-2(mMLC-2a),human atrial MLC-2a (hMLC-2a),and human ventricular MLC-2(mMLC-2v)demonstrates high amino acid homology of zebrafish MLC-2with both the atrial and ventricular isoform of human and mouse MLC-2.Black boxes indicate identical residues.A predicted EF-hand calcium-binding domain of zMLC2is indicated with bars above the alignment,and the phosphorylation site at serine 19is marked by a red-encircled P.Exon boundaries are indicated.C and D,The splice site donor-site mutation (g 3a)at the exon 2–intron 3boundary of zmlc2(C)results in 3different cDNAs in tel mutant embryos (D).Amino acid translation derived from the codons is shown below the base readouts.The mutated base is marked by an arrow.Exonic bases are displayed in uppercase;intronic bases in lowercase.D,In comparison with wild-type (wt)zMLC-2,3different cDNA products were identified in tel m225mutant cDNA,all predicted to lead to premature termination of transla-tion at the indicated (asterisk)location.326Circulation Research August 4,2006line,19,20which drives expression of green fluorescent protein (GFP)under control of a 5.1-kb upstream region of zmlc-2,specific fluorescence is found in both the atrium and the ventricle (Figure 4G through 4I),confirming that in contrast to mammals,there is no chamber restriction of MLC-2in zebrafish.In addition,after searching the zebrafish genomic and EST databases and performing consecutive RNA expression anal-yses,an additional cardiac regulatory z mlc gene could not be identified,indicating that similar to flies 21and frogs,22ze-brafish express only 1regulatory mlc-2in the heart.tel Is Essential for Cardiac MyofibrillogenesisIn mammals,the structural and functional roles of cardiac regulatory MLCs are still debated.In contrast to zebrafish,2regulatory MLC isoforms (MLC-2a and MLC-2v)arecoex-Figure 3.Inhibition of zmlc-2function by morpholino-modified antisense oligonucle-otide injection phenocopies the tel mutant phenotype.A through C,Ninety-seven percent of wild-type embryos injected with MO-mlc-2,which is directed against the translational start site of zmlc-2,are indis-tinguishable from tel mutant embryos and display severe impairment of cardiac con-tractility.D,Injection of BAC159f11DNA into tel Ϫ/Ϫmutant embryos rescues the tel mutant heart phenotype in 25%.Similarly,the tel mutant heart phenotype can be suppressed by injection of wild-typezmlc-2RNA (capped mRNA wt),whereas injection of tel mutant cDNA-1(capped mRNA mt)in tel Ϫ/Ϫmutant embryos had no effect on the cardiacphenotype.Figure 4.tel mutant hearts do notexpress truncated zMLC-2,whereas,in wild-type (wt)hearts,zmlc-2isexpressed in both heart chambers.A and B,By 72hpf,only traces of zmlc-2are detectable in tel mutant hearts com-pared with wild-type by whole-mount antisense in situ hybridization.C,In vitro translation and immunoblotting ofC-terminal (lane 1and 2)or N-terminal (lane 3and 4)FLAG-tagged wild-type (lane 1and 3)or mutant (lane 2and 4)zmlc-2.The arrow at 20kDa indicates wild-type zmlc-2(lanes 1and 3).The arrowhead indicates the expectedmolecular mass (in kilodaltons)of mutant zMLC-2,which is not expressed at detectable levels (lanes 2and 4).D through F,zmlc-2RNA is expressed in both cardiac chambers,atrium (A)and ventricle (V),during embryonic develop-ment (D and E)and in the adult zebrafish heart (F).G through I,Our transgenic zebrafish line Tg(cmlc2:GFP)expressing GPF under control of the zmlc-2pro-moter confirms transmyocardial expres-sion of zMLC-2.Tg(cmlc2:GFP)embryos show heart-restricted GFP expression in both chambers during embryonic devel-opment (G and H)and in the adultzebrafish heart (I).G and H,Frontal view;I,lateral view of an adult heart of aTg(cmlc2:GFP)zebrafish after removal of the gill cover (opercle).Rottbauer et al Cardiac Myosin Light Chain-2in Zebrafish Heart 327pressed in the heart and seem to partially compensate for the loss of the other MLC-2at least in the ventricle.23Interest-ingly,knockout mice recently revealed an essential role of MLC-2a in myofibrillogenesis of atrial cardiomyocytes.24Therefore,to determine whether the tel mutation renders ventricular and atrial cardiomyocytes noncontractile because of defective myofibrillogenesis,we analyzed the ultrastruc-ture of tel embryonic hearts by transmission electron micros-copy (Figure 5A through 5F).Normally,by 24hpf,nascent myofibrillar arrays can be observed in wild-type zebrafish hearts,and by 48hpf,clear organization of thick and thin filaments with discernible A-,I-,and Z-bands is evident (Figure 5A and 5C).In tel homozygous mutant hearts,organized thick and thin filaments are completely absent,both in the ventricle and atrium (Figure 5B,5D,and 5F).Only an immature assembly of thin-filament actin (Z-bodies)can be observed.As opposed to the tight hexagonal lattices of thick and thin filaments seen in cross-sections of wild-type myofibrils,only scattered thin filaments are present in tel mutant cardiomyocytes (Figure 5E and 5F,insets).Because,as outlined above,only cardiomyocytes of the AV canal contract in tel hearts,we examined whether these cardiomyocytes,in contrast to those of the ventricle and the atrium,form regular sarcomeres.However,our detailed ultrastructural analysis of AV cardiomyocytes from tel hearts also did not reveal any organized sarcomeric structures (data not shown).This indicates that even a loose interaction ofactin and myosin filaments in myocardial cells of the AV canal suffices to maintain their contractility.To examine whether altered gene expression of key regu-latory AV canal markers accounts for the changed morphol-ogy but preserved contractile function of the AV canal in tel mutant hearts,we performed whole-mount RNA expression studies for versican,an extracellular matrix protein,and bone morphogenetic protein 4(bmp4),both of which are known to localize to the AV-border during zebrafish heart development to control AV canal development.25,26However,both versi-can and bmp4are found to be expressed at normal levels and in a rather restricted expression pattern at the AV canal in tel mutant embryo,indicating that altered expression of neither versican nor bmp4causes the altered AV canal morphology in tel mutant embryos (Figure 6A through 6F).Cardiac Thick-and Thin-Filament Components Are Expressed at Normal Levels in tel Mutant HeartsSimilar to tel mutant hearts,loss of zebrafish troponin T also inhibits cardiac myofibrillogenesis.27Interestingly,troponin T deficiency also leads to significantly reduced expression of other thin-filament components,such as ␣-tropomyosin and troponin I,indicating interdependence of thin-filamentpro-Figure 5.tel is essential for thick-filament assembly in thezebrafish heart.A through F,Transverse sections of myocardial cells from the ventricular chamber are shown by transmission electron microscopy at different developmental stages (A and B).Whereas in wild-type (wt)ventricular cardiomyocytes,thick and thin filaments are already organized in sarcomeres,with discernible Z-discs,A-bands,and I-bands (A,C,and E),in tel m225Ϫ/Ϫcardiomyocytes,no higher-order sarcomeres can be detected (B,C,and D).Only an immature assembly of thin-filament actin (Z-bodies)can be observed in tel mutant cardio-myocytes.As opposed to the hexagonal lattices of thick and thin filaments seen in cross-sections of wild-type myofibrils (E,inset),only scattered thin filaments are present in tel mutant cardiomyocytes (F,inset).Figure 6.Abnormal AV canal morphology in tel mutant embryos is not attributable to altered expression of AV canal genes versi-can and bmp-4.A and B,The AV canal (av)interconnecting the atrial (A)and ventricular (V)chamber appears rather extended in tel mutant hearts (B)and maintains its teral view of a wild-type (wt)(A)and tel mutant (B)heart after 72hpf.C through F,Expression of the extracellular matrix protein versican and of Bmp4,which are known to localize to the AV border dur-ing zebrafish heart development,is at normal levels and in its restricted pattern in wild-type and tel mutant hearts.C through F,Frontal view of wild-type (C and E)and tel mutant (D and F)zebrafish embryos after RNA antisense in situ hybridization with a riboprobe directed against zebrafish versican (C and D)or bmp4(E and F).it indicates inflow tract;ot,outflow tract.328Circulation Research August 4,2006tein expression in the zebrafish heart.27Therefore,to further investigate whether absence of zMLC-2in tel mutant hearts also leads to reduced expression of other sarcomeric proteins and thereby accounts for disturbed myofibrillogenesis,we examined the expression of various cardiac sarcomeric pro-teins by in situ hybridization and immunostaining of tel mutant embryos.However,neither essential components of heart muscle thick filaments such as zebrafish atrial myosin heavy chain (zamhc )(Figure 7A and 7B)and zebrafish ventricular myosin heavy chain (zvmhc )(Figure 7C through 7E)nor thin-filament components such as ␣-tropomyosin (␣-TM)are found to be expressed at reduced mRNA and/or protein levels throughout tel mutant hearts (Figure 7G and 7H).Thus,in contrast to zebrafish troponin T,loss of zebrafish MLC-2does not lead to reduced expression of other thick-or thin-filament sarcomeric proteins,indicating that zMLC-2by itself is essential for cardiac myofibrillogenesis.DiscussionThe early and rapid nature of cardiac myofibrillogenesis poses challenges for in vivo studies.Assessing early embry-onic heart morphology and function is facilitated in zebrafish embryos,because they are transparent and not dependent on intact cardiovascular function during the first 7days of development.28Here we show,for the first time,that MLC-2is essential for thick-filament assembly and myofibrillogenesis in the zebrafish embryonic ing a forward genetic approach,we identified a recessive,embryonic lethal mutation in the zebrafish cardiac myosin light chain 2gene,tell tale heart.The mutation completely abolishes MLC-2function,result-ing in disturbance of myofibrillar assembly in a cell-autonomous fashion in tel mutant hearts.Consecutively,atrial and ventricular cardiomyocytes are unable to contract.Despite major advances in understanding the molecular basis of active contraction,surprisingly little is known about the mechanisms controlling myofibrillar formation in cardiac muscle.During early cardiac myofibrillogenesis,first I-Z-I complexes (Z-bodies)containing ␣-actinin,sarcomeric actin,and the amino terminus of titin are build in register.Thick filaments assemble and then incorporate into these preformed structures.Like other myofibrillar constituents,myosin first appears diffusely in the ter,when M-line epitopes of titin appear,myosin thick filaments organize in a sarcomeric arrangement together with I-Z-I complexes.How-ever,the exact mechanisms by which vertebrate sarcomeric myosin assembles into thick filaments are unknown.29We show here,for the first time,that MLC-2is essential for cardiac thick-filament assembly in vivo.In agreement with previous reports that thick and thin filaments assemble independently,30initial thin-filament assembly (formation of I-Z-I bodies)proceeds normally in tel mutant cardiomyo-cytes.However,in the absence of tel (MLC-2),thick-filament assembly is completely disrupted and organized sarcomeric units cannot be observed.This is despite the fact that cardiac myosin heavy chain is expressed at normal levels in tel mutant hearts,implicating an essential role of regulatory MLCs in thick-filament assembly and sarcomerogenesis.Interestingly,loss of titin in zebrafish leads to similar alter-ations in cardiac ultrastructure,15implicating potential over-lapping functions of MLC-2and titin in regulating cardiac myofibrillogenesis.In contrast to mammals,only 1mlc-2gene is expressed in the zebrafish heart.Similarly,only 1regulatory MLC gene exists in flies.Loss of mlc-2in Drosophila melanogaster is recessive lethal and leads to a dominant flightless phenotype.Even indirect flight-muscle myofibrils from heterozygotes are aberrant,exhibiting myofilaments in disarray at the periphery.These results indicate that wild-type MLC-2stoi-chiometry is required for normal flight-muscle assembly and function in flies.21Interestingly,heterozygous tel mutant embryos do not display an obvious cardiac phenotype.However,future studies of the heart morphology and function of adult heterozygous tel fish are necessary to examine the effect of heterozygous loss of zMLC on cardiac myofibrillo-genesis in the adult heart.The physiological in vivo function of regulatory MLCs in the vertebrate heart is debated.In contrast to zebrafish and flies,2major cardiac MLC-2isoforms are coexpressed in the early stages of mouse cardiogenesis,a ventricular (MLC-2v)and an atrial isoform (MLC-2a).31Both isoforms seem to be able to partially compensate for the loss of the other MLC-2.Disruption of the ventricular myosin light chain 2gene (mlc-2v )in mice is lethal at embryonic day 12.5as a resultofFigure 7.Cardiac thick-and thin-filament components areexpressed at normal levels in tel mutant hearts.A through D,By 72hpf,the expression of atrial myosin heavy chain (amhc )(A and B)and ventricular myosin heavy chain (vmhc ),investigated by RNA antisense in situ hybridization,is similar in tel (B and D)and wild-type (A and C)hearts.E and F,Normal and chamber-restricted expression of cardiac myosin proteins (E and F)and ␣-tropomyosin (␣-Tm)(G and H).Lateral view of wild-type and tel mutant embryos at 48hpf.MF20(TRITC)stains the entire heart tube;S46(FITC)stains only the atrium.In double expo-sure,the ventricle (V)fluoresces red and the atrium (A)fluo-resces yellow.Rottbauer et al Cardiac Myosin Light Chain-2in Zebrafish Heart 329。
