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浙江大学文本 Replacement of Connexin40 by Connexin45 in the Mouse（cbp）_生理学

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Replacement of Connexin40 by Connexin45 in the Mouse Impact on Cardiac Electrical Conduction Sehastien Alcolea,Therese Jarry-Guichard,Jacques de Bakker,Daniel Gonzalez,Wouter Lamers, Steven Coppen.Luis Barrio.Habo Jongsma,Danicl Gros.Harold van Rijen Astro-Gap juction channels required for the propagntion of cardiac impulse,are intercellular structures composed of cunnexins (Cx).Cx13.Cxlo,and Cxl5 are synthesized in the cardiomyocytes,and each of them has a unique cardisc expressiun pattem Cx40 knock in Cx45 mice were generated lu explore the ability of Cx45 to replabe Cx40,and to assess the functional equrvalerce of these two Cxs that are both expressed in the conduction system.ECs revealed that the corsequences resulting from the hinllelic replacement of Cx40 by Cx45 were an increased durnticn of the P wave, and a prolonged and fractienaled QRS complex.Epicardial mapping indicated that the cenduction velocities(CV)in the righ atrum and the venmeular myocardium,ns well as eonduction throngh the Av node,were unaffected.The significant reduction ofthe CV in the lett atrium woud be the most likely cause of the P-wave lengthening.In the right ventricle,a changed and prolonged nctivation in sinus rhythm wns tound in homozygous mutant mice,which may explain the prulengation ad splitting of the QRS complex.Electrical mapping of the llis bundle branches revealed that this wns due to slow conduction measurod in the right bennch The CV in the lett branch wns unchneed.Therctore.in the absence of Cx40,the upregulation of Cx45 in the heart results in a noemal impulse propagation in the right atrium, the AV node.and the left His bundle brarch only.[Cire Res.2004;94:100-109.) Key Words:gnp junction channels connexins cardine conduction In the mimmalian heart the cletneal aetivity,spontine- imesligtioe Lastly,Cx wis deteeted in the rabhit SA oy pencaled in the sin81l(代A),s国eh me igocinted with C40 and Cx45 through the atna,then to the ventricular walls via specialized Analysis of Cx-deficient mice has provided evidence that tissues that constinute the conduction sysiem [(S).The CS (x43.Cx40,and Cx45 are involved in both heart function includes the atrioventriculr(AV)node.the His bundle,ad and development.The Cx45 rull mutatio is lethal with the the two bundle branches(U).whoch ramify inn peripheral embryos dying i uero around embrvyocic day 10.2 dzys Purkinje fibers.Gap junction channels are responsible for the afkr cardiac cornictions are nemally nlod 136 Several electrical coupling between the various types of myocyles. anomalies including defects in the endocardal cushicns are and they are required for the propegation of'csndiae impulse thoght to comrute to the lethal phenutype.Conditional Gp union chuneb ae itteellular snlures xe kr-u (KO)mice in which Ca43 genc expession in The tied by transmembeare peoteirs belonging to the connexin cardiomyocytes is reduced by about 90%have ventricular (Cx)fimily .Nineteen Cx genes have been idertifled in the candaction velocity (CV)slowed by 50%.All these mice mouse genoee,but so far only four Cxs,Col3.Cx0,C5. udergo sudden cardiac death from spontaneous ventricular and Cx46,have been shown to be expressed in mammalian arhythmias by 2 moeths of ageCxko mice are viabke crdiumyoryles with cach hving3miqp代3sin图en an时fartile,md several suis ve fiocd on impuls买 in the adult heart Hence.in the mouse beart.Cx43 is rup四gation in the hearts of thes0miee2-达Findings from abundan:in both the atrial and ventricular working myocytes, these stodies include impaired conduction at various levels of and the hsbl putofthe C5 Cxo is srongby repreal in the the ('S.and incteased incienc of inducible urial alrisl wurking myocytes and the CS.whereas Cx45 is achyarrinthis,indicaling lhat Cx40 is an importanl doer- detected mainly n the SA node and the CS.Expression of minant of impulse propagation in the atria and the Av Cx45 has been repored in the werking vencneur myoer- c0nd,n对sem民oc女lin吧mml3 suggest that diam burt this finding has not been corrobarated by other Cx40 may ply a role in cardiac morphogecesis. Oitgital ovcived May 12,2003,Beunosio iee:ivod October 2,2003,nevbed cosubnbsion ooeved Nowenber 6,2003;aoxped Noveseer 11. 20E. Igr程he Lahorarore de《enengu6 nsolosie du门elepperent(LUR%Hs5升区A,T果i_DGL legnit de lolos au nlevekrpen㎡ert Netherands.Neurolga Expenmertal DG L.B.1.Hospital Ranon y Cajl Modnd,Spain.Imeruriversty Carcidlogy Iroutue (dB.)Uhech.The ig8业iactmmfmnnnzNhtnk Cuequsdcie D Daricl Geea LGPDIIBDM,Canp de Lunary,Cow 927,13288 Mat:lle,Fuke.E-til suibent.uiv-fi C 2004 American Heant Axorcitioe,he. Cireweda Reseth is的lable ar bit民w4wirt代sahaarg D0E1011610L.RE504018616992A 100

Replacement of Connexin40 by Connexin45 in the Mouse Impact on Cardiac Electrical Conduction Sébastien Alcoléa, Thérèse Jarry-Guichard, Jacques de Bakker, Daniel Gonzàlez, Wouter Lamers, Steven Coppen, Luis Barrio, Habo Jongsma, Daniel Gros, Harold van Rijen Abstract—Gap junction channels, required for the propagation of cardiac impulse, are intercellular structures composed of connexins (Cx). Cx43, Cx40, and Cx45 are synthesized in the cardiomyocytes, and each of them has a unique cardiac expression pattern. Cx40 knock-in Cx45 mice were generated to explore the ability of Cx45 to replace Cx40, and to assess the functional equivalence of these two Cxs that are both expressed in the conduction system. ECGs revealed that the consequences resulting from the biallelic replacement of Cx40 by Cx45 were an increased duration of the P wave, and a prolonged and fractionated QRS complex. Epicardial mapping indicated that the conduction velocities (CV) in the right atrium and the ventricular myocardium, as well as conduction through the AV node, were unaffected. The significant reduction of the CV in the left atrium would be the most likely cause of the P-wave lengthening. In the right ventricle, a changed and prolonged activation in sinus rhythm was found in homozygous mutant mice, which may explain the prolongation and splitting of the QRS complex. Electrical mapping of the His bundle branches revealed that this was due to slow conduction measured in the right branch. The CV in the left branch was unchanged. Therefore, in the absence of Cx40, the upregulation of Cx45 in the heart results in a normal impulse propagation in the right atrium, the AV node, and the left His bundle branch only. (Circ Res. 2004;94:100-109.) Key Words: gap junction channels connexins cardiac conduction I n the mammalian heart the electrical activity, spontaneously generated in the sinoatrial (SA) node, is propagated through the atria, then to the ventricular walls via specialized tissues that constitute the conduction system (CS). The CS includes the atrioventricular (AV) node, the His bundle, and the two bundle branches (BBs), which ramify into peripheral Purkinje fibers. Gap junction channels are responsible for the electrical coupling between the various types of myocytes, and they are required for the propagation of cardiac impulse.1 Gap junction channels are intercellular structures constituted by transmembrane proteins belonging to the connexin (Cx) family.2,3 Nineteen Cx genes have been identified in the mouse genome,4 but so far only four Cxs, Cx43, Cx40, Cx45, and Cx46, have been shown to be expressed in mammalian cardiomyocytes with each having a unique expression pattern in the adult heart.5 Hence, in the mouse heart, Cx43 is abundant in both the atrial and ventricular working myocytes, and the distal part of the CS; Cx40 is strongly expressed in the atrial working myocytes and the CS,6,7 whereas Cx45 is detected mainly in the SA node and the CS.8–10 Expression of Cx45 has been reported in the working ventricular myocardium11 but this finding has not been corroborated by other investigations.9,12 Lastly, Cx46 was detected in the rabbit SA node associated with Cx40 and Cx45.13,14 Analysis of Cx-deficient mice has provided evidence that Cx43, Cx40, and Cx45 are involved in both heart function and development. The Cx45 null mutation is lethal with the embryos dying in utero around embryonic day 10, 2 days after cardiac contractions are normally initiated.15,16 Several anomalies including defects in the endocardial cushions are thought to contribute to the lethal phenotype. Conditional knock-out (KO) mice in which Cx43 gene expression in the cardiomyocytes is reduced by about 90% have ventricular conduction velocity (CV) slowed by 50%. All these mice undergo sudden cardiac death from spontaneous ventricular arrhythmias by 2 months of age.17 Cx40KO mice are viable and fertile,18,19 and several studies have focused on impulse propagation in the hearts of these mice.20–25 Findings from these studies include impaired conduction at various levels of the CS, and increased incidence of inducible atrial tachyarrhythmias, indicating that Cx40 is an important determinant of impulse propagation in the atria and the AV conduction system. Recent investigations also suggest that Cx40 may play a role in cardiac morphogenesis.26 Original received May 12, 2003; resubmission received October 2, 2003; revised resubmission received November 6, 2003; accepted November 11, 2003. From the Laboratoire de Génétique et Physiologie du Développement (UMR CNRS 6545) (S.A., T.J.-G., D.G.), Institut de Biologie du Développement de Marseille, Université de la Méditerranée, Marseille, France; Department of Medical Physiology (H.v.R., H.J.), University Medical Center, Utrecht, The Netherlands; Neurologia Experimental (D.G., L.B.), Hospital Ramón y Cajal, Madrid, Spain; Interuniversity Cardiology Institute (J.d.B.), Utrecht, The Netherlands; Department of Anatomy and Embryology (W.L.), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands, and National Heart and Lung Institute (S.C.), Imperial College London, UK. Correspondence to Dr Daniel Gros, LGPD/IBDM, Campus de Luminy, Case 907, 13288 Marseille, France. E-mail gros@ibdm.univ-mrs.fr © 2004 American Heart Association, Inc. Circulation Research is available at http://www.circresaha.org DOI: 10.1161/01.RES.0000108261.67979.2A 100

102 Circulation Research January 9/23,2004 KI/KI Cx40 Figure 2 Expression pettem of Cus asso dated with myocytes in the stria af WT ++ +/+ KIK图 micmgraph of freoer aAdtion from atria of Cx40(-/+)and Cx40 129SC5781/6 mice [K/KI Soctian wero treaed with rabbit anti-Cx40 (A and B). 南-x45end米.cfrh-Cx43LrG F)antibodios.A,B'.C,D.E,and F,Im muronuorescencc micrographs B,C',and C 、45 shoan in B.C and F',respectrely.Smi- KI/KI mntA.日e-20 m for A thm好中FF Th anulysis ol'KO mioe fas thareloe providod important euring epicardial octivoion and山e conduction vdocity (CV门in information on the in vivo functions of Cxs in heart.Another the various candiac tssues ncludig the BUs are describod in the approach for analyzing the function of Cxs is to generate online dats supplerent.Multiple group compansors were performed sinz ANOVA with LSD post loe analysis iang SPS5 10 fior knock-in (KI)mice to assess the functional equvalence of Mocatos.Values are memntSEM.Vaues of P-005 were cou- two Cxs.Cx40 and Cx45 are both expressed in the mouse CS sdce对tatistically signfic3m albeit with very diferen levels of expresson Chanrels compoeed of Cx40 or Cx45 have quite different properties Results Cx40 channels have a large unitary conductance (160 pS) in contrast with Cx45 charmels,which have a small conduc- Generation of the KI Mice tance (-30 pS)Macrosoopic conductance of Cx40 chan KI mice were generated on two genctic backgrounds:129129/ els is not very sensitive to the tran可junctoral volt买V,)as CD1(50%50%)d129sC571崦(50/%/50%).No abn0r compared with that of Cx45 charrels,which is very sensitive mal embryonie lethality was detecol The mice grew nor- to VFurthermore,both types of channels are differently mally,were fortile.and had no obvious analomical cardiac regulalod by phospborylalion To assess the functionsl defocts. equivalence of Cx40 and Cx45,we have generated Cx40KICx45 mouse lines in which Cx45 is expressed in lieu Transeriptional and Translational Expressions of and place of Cx40. the Transgenc The results revealed no significant difference deperdent on Materials and Methods the genctic buckyround and were reproducible for each type The stralegy u%d切rc红ate the tramspenc mice and the tochniu used for penutyping the Es oells and mice and for identifying of experiment perfurmed. expression of the transgene in the KI mioe are descnbed in detari in The expression of Cx40.Cx43,ad transgenic Cxl5 the expended Miteials ad Methods in the osline dotn supplemet mRNAs in the atria of wild-type (wt,Cx4)heterozygous (availabl at ltp:0www.eacreshn org)The expeession ot'the trs- (Cx40)and homnzygous (Cx4)mice was ae was aceced on three herrts foa adut mce (8 to 12 weeks determined by smiquantilalive RT-PCR (Figure 1B)and old),from three independent littes,for each type of experimeat camied out (RT-PCR.Northem and Wesem blos iamusofluoces- Nocthern blot experimerts (Figure IC)No endogenous Cx45 cenee)and fur cach ohpe imrstirsied In a:dtin.all exeri- expressicn wus detecied (Figures IB and IC but a gene ments were perforemed with smples from both Sv129CD)I and dependent gain of Cx45 mRNA.accompanied by a gene 128￥C576Hx. dpender loss of Cx40 tnmscript (Figures IB and IC)was FC0 neordings were performted on anesthetired mire深雀- sribd previmsdy The hearts wer dissected,comed toa 3nmCx40*r“and Cx40rei“mic.The expe3ew Langendorif setup,ind extracelalar epicerdid electrogroms were level ofCx45 mRNA in Cx mice was coenparable ecorded daring sirus (o puced rhythm.Protocols fur to that of Cx40 mRNA in Cx4omice (Figures IB and IC)

The analysis of KO mice has therefore provided important information on the in vivo functions of Cxs in heart. Another approach for analyzing the function of Cxs is to generate knock-in (KI) mice to assess the functional equivalence of two Cxs. Cx40 and Cx45 are both expressed in the mouse CS, albeit with very different levels of expression. Channels composed of Cx40 or Cx45 have quite different properties. Cx40 channels have a large unitary conductance (160 pS), in contrast with Cx45 channels, which have a small conductance (30 pS).27,28 Macroscopic conductance of Cx40 channels is not very sensitive to the transjunctional voltage (Vj) as compared with that of Cx45 channels, which is very sensitive to Vj. 29–32 Furthermore, both types of channels are differently regulated by phosphorylation.28,33 To assess the functional equivalence of Cx40 and Cx45, we have generated Cx40KICx45 mouse lines in which Cx45 is expressed in lieu and place of Cx40. Materials and Methods The strategy used to generate the transgenic mice and the techniques used for genotyping the ES cells and mice and for identifying expression of the transgene in the KI mice are described in detail in the expanded Materials and Methods in the online data supplement (available at http://www.circresaha.org). The expression of the transgene was assessed on three hearts from adult mice (8 to 12 weeks old), from three independent litters, for each type of experiment carried out (RT-PCR, Northern and Western blots, immunofluorescence), and for each genotype investigated. In addition, all experiments were performed with samples from both Sv129/CD1 and 129Sv/C57Bl/6 mice. ECG recordings were performed on anesthetized mice as described previously.25,34 The hearts were dissected, connected to a Langendorff setup, and extracellular epicardial electrograms were recorded during sinus (SR) or paced rhythm.25,34 Protocols for measuring epicardial activation and the conduction velocity (CV) in the various cardiac tissues including the BBs are described in the online data supplement. Multiple group comparisons were performed using ANOVA with LSD post hoc analysis using SPSS 10 for Macintosh. Values are meanSEM. Values of P0.05 were considered statistically significant. Results Generation of the KI Mice KI mice were generated on two genetic backgrounds:129129/ CD1 (50%/50%) and 129Sv/C57Bl/6 (50%/50%). No abnormal embryonic lethality was detected. The mice grew normally, were fertile, and had no obvious anatomical cardiac defects. Transcriptional and Translational Expressions of the Transgene The results revealed no significant difference dependent on the genetic background and were reproducible for each type of experiment performed. The expression of Cx40, Cx43, and transgenic Cx45 mRNAs in the atria of wild-type (wt, Cx40/), heterozygous (Cx40/KICx45) and homozygous (Cx40KICx45/KICx45) mice was determined by semiquantitative RT-PCR (Figure 1B) and Northern blot experiments (Figure 1C). No endogenous Cx45 expression was detected (Figures 1B and 1C), but a genedependent gain of Cx45 mRNA, accompanied by a genedependent loss of Cx40 transcript (Figures 1B and 1C) was seen in Cx40/KICx45 and Cx40KICx45/KICx45 mice. The expression level of Cx45 mRNA in Cx40KICx45/KICx45 mice was comparable to that of Cx40 mRNA in Cx40/ mice (Figures 1B and 1C). Figure 2. Expression pattern of Cxs associated with myocytes in the atria of WT and transgenic adult mice. Shown are micrographs of frozen sections from atria of Cx40/ (/) and Cx40KICx45/KICx45 129Sv/C57Bl/6 mice (KI/KI). Sections were treated with rabbit anti-Cx40 (A and B’), anti-Cx45 (C and D), or anti-Cx43 (E and F’) antibodies. A, B’, C, D, E, and F’, Immunofluorescence micrographs. B, C’, and F are Nomarski micrographs of sections shown in B’, C, and F’, respectively. Similar results were obtained with 129Sv/CD1 mutant mice. Bar20 m for A through F’. 102 Circulation Research January 9/23, 2004

Alcolia et al Comnexin40 Knock-In Comnexin45 Mice 103 Cs +/+ LVC Cx40 Cx45 KI/KI KIKI Cx45 aEpc2onaemHcesese黑93A5n298e9ga"8mnd eraph&of Iran saction&Imm the vamricle al Cx40/)A.D.E.Are F)and Cx40 Sectiors warn traned with both rabit anti-Cx40 antibodas (Cx40),and g_inea pig anti-Ce45 Cx45 (doubla-immuno- Huoresoence technique Supermposed Nomarsk and immunotuorescence micrograohs of sections af coronsry wessels in the cardiac wall are preented nA and .mmoonndmed betwenhe endatholial oalls of coronarios of Cx40"'mice A groon signal has boon replocod by C45 in Cx40 nice B.red signa出.D and G are Nomarski micrographs.on indicales endocardium IVS.septum:and LVC.lelt ventncular chamter.E.F.and Hare immunolluoreecenc#meo灯aphs Pasiton ofth对ssca8中cen in the8 s paneis8 ndcaled by8 in gcher时Ct中rsp hnf8Gx0+m网%.Exprexpsion ol bath Ca84tT8nH线oundle brsnch,目WkGx5lr时株8v6nh州 vicirity of the in the NvS (amws in P In the Cx mice (C and H.as in the Cx45 was in the His bundle branches twhte amow in Hl,and in the first layer cels of the septal working myocytes [black amows in Hi.was undetect- a止e in the major poron of the ptum laster in H.Smiar reace um for A and B.her in D-50 um for D.E,and F:and bar in G-20 jumm for 3 and H. The levels of expression of the transcript of the Cx43 gere in with a level of expression higher than that observed in the the three types of'mmice imestgated were similar (Figure IC) heterozygoes.The level of expression of Cx43 protein was Cx45 protein was correctly translated in the transgenic unaffected by the expression of the trarsgene (Figure ID) mice.as shown by Weskern blol experiments perfurmod with The puller of expressiun of Cx40.Cx45,and Cx43 was atrial samples (Figure 1D)The reduction of the level of further irvestigated hy immunofluorescence on sections of expression of Cx40 protein in the ar of Cx mice Cx4 and Cx4 mouse beart No signal was (Figure ID)wns associated with the derection of Cx45 detected in the atria of Cx4 mice treated with protein in the same tissue.Cx40 procein was not detected in anti-Cx40 antibodies (Figure 2B').or in the atri of Cx40 the atrin samples of C4 miee In contrest.Cx45 mice treaed with ant-Cx45 smibodies (Figure 2C)In protein was shown to be expressed in these same samples contrast,analysis of sections from atria of Cx40

The levels of expression of the transcript of the Cx43 gene in the three types of mice investigated were similar (Figure 1C). Cx45 protein12 was correctly translated in the transgenic mice, as shown by Western blot experiments performed with atrial samples (Figure 1D). The reduction of the level of expression of Cx40 protein in the atria of Cx40/KICx45 mice (Figure 1D) was associated with the detection of Cx45 protein in the same tissue. Cx40 protein was not detected in the atria samples of Cx40KICx45/KICx45 mice. In contrast, Cx45 protein was shown to be expressed in these same samples with a level of expression higher than that observed in the heterozygotes. The level of expression of Cx43 protein was unaffected by the expression of the transgene (Figure 1D). The pattern of expression of Cx40, Cx45, and Cx43 was further investigated by immunofluorescence on sections of Cx40/ and Cx40KICx45/KICx45 mouse heart. No signal was detected in the atria of Cx40KICx45/KICx45 mice treated with anti-Cx40 antibodies (Figure 2B’), or in the atria of Cx40/ mice treated with anti-Cx45 antibodies (Figure 2C). In contrast, analysis of sections from atria of Cx40KICx45/KICx45 Figure 3. Expression pattern of Cxs associated with myocytes in the ventricles of WT and transgenic adult mice. Shown are micrographs of frozen sections from the ventricles of Cx40/ (/) (A, D, E, and F) and Cx40KICx45/KICx45 129Sv/CD1 mice (KI/KI) (B, G, and H). Sections were treated with both rabbit anti-Cx40 antibodies (Cx40), and guinea pig anti-Cx45 antibodies (Cx45) (double-immuno- fluorescence technique). Superimposed Nomarski and immunofluorescence micrographs of sections of coronary vessels in the cardiac wall are presented in A and B. ec indicates endothelial cells; sm, smooth muscle cells; and m, myocytes. Cx40 detected between the endothelial cells of coronaries of Cx40/ mice (A, green signals) has been replaced by Cx45 in Cx40KICx45/KICx45 mice (B, red signals). D and G are Nomarski micrographs. en indicates endocardium; IVS, interventricular septum; and LVC, left ventricular chamber. E, F, and H are immunofluorescence micrographs. Position of the sections shown in these panels is indicated by a box in scheme C, which represents a mammalian heart. E and F, Subendocardial expression of Cx40 and Cx45, respectively, in a section (D) coming from the heart of a Cx40/ mouse. Expression of both Cxs overlaps in the His bundle branch, but weak Cx45 labeling is also observed in the vicinity of the bundle, in the IVS (arrows in F). In the Cx40KICx45/KICx45 mice (G and H), as in the Cx40/mice, Cx45 was detected in the His bundle branches (white arrow in H), and in the first layer cells of the septal working myocytes (black arrows in H). It was undetectable in the major portion of the septum (asterisks in H). Similar results were obtained with 129Sv/C57Bl/6 mutant mice. Bar in A50 m for A and B; bar in D50 m for D, E, and F; and bar in G20 m for G and H. Alcoléa et al Connexin40 Knock-In Connexin45 Mice 103

I04 Circwlalion Research Janry9/23,2004 C57H6 mice were similar to thase of the 1298v/CD)1 mice (not shown)However the absoline values of the measured parnmeters were different from those of 129SvCDI miee C40+- (Table)indicating a strain dependency,as expected.The P wave and the ORS complex durationss were significantly increased in the Cx40 129Sv/C57Bl/6 mice,a compared with the Cxomice.Beca.sse the modificaticns identified were similr in the two geretic backgrounds,the experiments that followed were perfommed on one back- ground only.129Sv/CDI. Ventricwlar Acfiration Epicardiul mapping was perfotmed in SR un Langenkor Cx04n perfused hearts to imestigate the delayed activation of the ventricles in Cx mice Figure shows represen- tstive activation maps of the right (Figure 5A)and left 20 ms ventricles (Figure 5B)in SR In the Cx40and Cx40*s mice,the sites of first activation were found as breakthrough Figure 4.Repreocntathe ECGo rocorded tom Cx40 apiculateral.or biclcrul sites (sa Table for the frcyuencics Cx and Cx40 mioe.Genetic background, 1288wCD1.Prlergstion and frastionstion ofth ORS complex of events).In thCx mice no midventricular was observed in the C40 mice.Dotted lnes represent hrenkthrough nctivation wns found,and the earliest activa- isceleotrio lines. tions were predominantly found at apical sites (Tahle). indicating a different mode of ventricular activation in these mie indicalad that Cx45 was sronghy expressal in this lissue anmaks. with a pattem of'expression similar to tht of Cx40 in the The tocal activation time was calculaved from a RV and samples of Cx40"mice (compure Figures 2A and 2D)The LV activa减ion maps recorded in SR bry subtracting the lates过 pattern of expression of Cx43,and the intensiry of signals activation times from the earliest ones.The activation time of were similar in the atrin of both Cx4 and Cx the RV was significamtly increased from 54+04 ms in mice (Figures 2E and 2F).Cx40 and Cx45 proteins were Cx40 mice to 907 ms (67%)in Cx4 mice detected in the cardiac CS of Cx40""mice (Figures 3D and (Table)The adtivution times of the LV of the mice of the 3F),as reporlod peeviuusly.The area of Cx45 labeling was three genotypes investigated were no significantly different larger than that of Cx4o,coresponding to the so-called (Table) "exended conductioa system"and the intensity of signal was wenker than that detected for Cx40 protein (Fipures 3F Atrloventricular Condnction and 3F)In the Cx4mice the pattern of'expression Figure sC shows excamples of the AV delay as n function of of Cx45 delineated an extended CS similar to that seen in the the coupling interval of the premature stimulus (conduction Cx40 mice (Figure 3H)Cx40 was detected between the curves)No significant differences were devected between the endothelial cells of the coronanes imignting the cardiac wnlls three grougs of miee for neither the AV eflective refractory of the Cxlo"mice (Figure 3A)In the coronaries of 对(ERP,minmum AV dcly减infinikcly long Cxmice,Cx45 had replaced Cx40,expected coupling intenals (D)nor the time constant ()(Tablek (Figure 3B) Afriul Com山ciow Activation mapss of the paced right (KA)and left (LA)atria of Analysis of the Impulse Conduction in the Heart the three groups of mice imvestigated are shown in Figures 6A Surface ECGs and 6B.respectively.In the RA.the CV was 30.81.8 cm/s Figure 4 illustmtes the ECG recordings of the 129SwCDI for the Cx40"mice and was not significantly changed in the mice.The values of the parameters of ECGs and their Cx40u and Cx40KKMsoM mice (32.9=2.3 and statistical analysis are summarized in the Table.No differ- 30023 cm/s.respectively)(Table)In the LA.there was a ence wns found between the three genotypes investigated for significant roductin of the CV.fiom 31.5=1.6 cm/s in the RR oc the PO intervals.However.the P wave duration Cx0 mice to 243+22 cm/in Cx mice wus significantly increased from 9.10.3 ms in the Cx40+ me0I0.7±0.5 ms in the Cx4ud“mie.sugeesting Ventricwlar Cowduction skwer r aclivalion or other mecanisms such aa Examples of activation maps of the paced RV and LV are delayed activation in the interatral pathways or ectopic shown in Figures 6C and 6D),respectively RV and I.V activntion The latter hypothesis is unlikely because the activation maps showed anisotropic conduction independent P-wave morphology was similar for all three genorypes The of the genatype.Boch longtudinal and transversal CVs in the QRS complexes were fractionated and their duration was ventricles were similar in all three genotypes (Table). increased from 10.80.4 ms in the Cx40+mice to 14.00.6 Buadle Branck Conduc市on msn心eCx40radw”me,oninayed Examples of RBB and LBB activalion maps are shown in tiun of the vertricles.The profiles of the ECGs of 129Sv Figures 6E ard 6F.tespectively.RBB actnvation was always

mice indicated that Cx45 was strongly expressed in this tissue with a pattern of expression similar to that of Cx40 in the samples of Cx40/ mice (compare Figures 2A and 2D). The pattern of expression of Cx43, and the intensity of signals, were similar in the atria of both Cx40/ and Cx40KICx45/KICx45 mice (Figures 2E and 2F). Cx40 and Cx45 proteins were detected in the cardiac CS of Cx40/ mice (Figures 3E and 3F), as reported previously.5 The area of Cx45 labeling was larger than that of Cx40, corresponding to the so-called “extended conduction system,”9 and the intensity of signal was weaker than that detected for Cx40 protein (Figures 3E and 3F). In the Cx40KICx45/KICx45 mice the pattern of expression of Cx45 delineated an extended CS similar to that seen in the Cx40/ mice (Figure 3H). Cx40 was detected between the endothelial cells of the coronaries irrigating the cardiac walls of the Cx40/ mice (Figure 3A). In the coronaries of Cx40KICx45/KICx45 mice, Cx45 had replaced Cx40, as expected (Figure 3B). Analysis of the Impulse Conduction in the Heart Surface ECGs Figure 4 illustrates the ECG recordings of the 129Sv/CD1 mice. The values of the parameters of ECGs and their statistical analysis are summarized in the Table. No difference was found between the three genotypes investigated for the RR or the PQ intervals. However, the P wave duration was significantly increased from 9.10.3 ms in the Cx40/ mice to 10.70.5 ms in the Cx40KICx45/KICx45 mice, suggesting slower atrial activation or other mechanisms such as a delayed activation in the interatrial pathways or ectopic activation. The latter hypothesis is unlikely because the P-wave morphology was similar for all three genotypes. The QRS complexes were fractionated and their duration was increased from 10.80.4 ms in the Cx40/ mice to 14.00.6 ms in the Cx40KICx45/KICx45 mice, pointing to a delayed activation of the ventricles. The profiles of the ECGs of 129Sv/ C57Bl/6 mice were similar to those of the 129Sv/CD1 mice (not shown). However the absolute values of the measured parameters were different from those of 129Sv/CD1 mice (Table), indicating a strain dependency, as expected.35 The P wave and the QRS complex durations were significantly increased in the Cx40KICx45/KICx45 129Sv/C57Bl/6 mice, as compared with the Cx40/ mice. Because the modifications identified were similar in the two genetic backgrounds, the experiments that followed were performed on one background only, 129Sv/CD1. Ventricular Activation Epicardial mapping was performed in SR on Langendorffperfused hearts to investigate the delayed activation of the ventricles in Cx40KICx45/KICx45 mice. Figure 5 shows representative activation maps of the right (Figure 5A) and left ventricles (Figure 5B) in SR. In the Cx40/ and Cx40/KICx45 mice, the sites of first activation were found as breakthrough, apicolateral, or basolateral sites (see Table for the frequencies of events). In the Cx40KICx45/KICx45 mice no midventricular breakthrough activation was found, and the earliest activations were predominantly found at apical sites (Table), indicating a different mode of ventricular activation in these animals. The total activation time was calculated from all RV and LV activation maps recorded in SR by subtracting the latest activation times from the earliest ones. The activation time of the RV was significantly increased from 5.40.4 ms in Cx40/ mice to 90.7 ms (67%) in Cx40KICx45/KICx45 mice (Table). The activation times of the LV of the mice of the three genotypes investigated were not significantly different (Table). Atrioventricular Conduction Figure 5C shows examples of the AV delay as a function of the coupling interval of the premature stimulus (conduction curves). No significant differences were detected between the three groups of mice for neither the AV effective refractory period (ERP), the minimum AV delay at infinitely long coupling intervals (D ), nor the time constant () (Table). Atrial Conduction Activation maps of the paced right (RA) and left (LA) atria of the three groups of mice investigated are shown in Figures 6A and 6B, respectively. In the RA, the CV was 30.81.8 cm/s for the Cx40/ mice and was not significantly changed in the Cx40/KICx45 and Cx40KICx45/KICx45 mice (32.92.3 and 30.02.3 cm/s, respectively) (Table). In the LA, there was a significant reduction of the CV, from 31.51.6 cm/s in Cx40/ mice to 24.32.2 cm/s in Cx40KICx45/KICx45 mice. Ventricular Conduction Examples of activation maps of the paced RV and LV are shown in Figures 6C and 6D, respectively. RV and LV activation maps showed anisotropic conduction independent of the genotype. Both longitudinal and transversal CVs in the ventricles were similar in all three genotypes (Table). Bundle Branch Conduction Examples of RBB and LBB activation maps are shown in Figures 6E and 6F, respectively. RBB activation was always Figure 4. Representative ECGs recorded from Cx40/, Cx40/KICx45, and Cx40KICx45/KICx45 mice. Genetic background, 129Sv/CD1. Prolongation and fractionation of the QRS complex was observed in the Cx40KICx45/KICx45 mice. Dotted lines represent isoelectric lines. 104 Circulation Research January 9/23, 2004

Alcolia et al Connexin40 Knock-In Connexin45 Mice 105 Electrephyslological Pamameters af Wild-Type and Mutant Mice 0404 40g CoGA0C ECG4wM8re门283a01 图Tsw 1285±11h-6 1225±a.1m-111 121±56作-10 07 P站3脑 9.1±033h-0 43±04=11l 107±05B=101 034 P ivd 34a+0.7-t4 344±07=11 354+11-10 063 s减n 10游±04们- 1g5±03-111 14±08=1W十 Q.30 C612 elrs me128wC57U阁 ft inavl 1043+5月=14转 1039+33=14 115+82他-1得 638 P特 94±0J0=10 1g5±02-1a 122±05m-1深十 o.c01 PO ilervdl 7±14=1号 3是.4±09=1a 42.1±19=1得 019 ORS duration 102±02们=14 105±03=1a 125±05n=1十 0.201 kia红r activetion2gwcD1 s特uu炉HwM % 1125喝 % ocad山 0%0 9%BN.% %g間 BxxMzienl RY LV) 17%17 0毫375为 3E%%] W actvason tin酸Ts 54±04m= 84±08= ±070=11t 0.4 LWxn1ne,形日.1±0月= 3.7±0.7=1 6.7±05n=14 Q.1 Conduction vclocitics,cm[129GWCD1] 队G 308±18们- 329±230-110 30.0±230-1☒ 063 IA CV 315+1月m-4 358+3.1=110 243+22m=111 61 fT/CV knjlueaed 375+27-t 331±8.7=1得 8±22m=10 05 RI CV tansversal 210±34你- 212±20-0 98±060-10 0.E0 形aniotmpic a 20+03 17+02 17+01 052 L Cr leogiatasl 4税3+42=有 38.1+32= 344+32m=1☑ 3约 LyGU克对 25±11-自 108士11-到 11±10m=1 011 LW修wt切 2J±03 22±02 21±02 Q.5 PBR CV 365+54m-59 4M8+5月=9 1绿2+29=11 0601 L倒C 4/8士7=斜 432士35-1 4明士58-图 Q粥 W rode fundon (1299vCD1) W邪，亚 6B3±4n=得 6因±5G=14 742±5.1n=14 0.53 州等一写移 557±25-t1 842±35-1W M9士241m-1☑ Q12 W手 J15±57们= 2Z13±1月=1 24.4±24m=1a Q14 hw路m除n球箱+SL.“5 comaee m Ce40r4t6H鲜dn合40e5 n indios nter sl ndependent cxporinerts recorded in a ooe-electrode wide strand,whereas LBB the involvemen.of Cx45 in the propagation of cardiac activation was electrically much wider and was alwnys impulse in the ahsence of Cx40 recorded in several cleetrode strands parallel to each other For both genctie bockgrounds investipied.the major In the Cx40'"mice.the CV in the RBB was 36.515.4 phcnoypic clfocts of'the ballcl teplacement of Cx40 by cm/s (Tablel.and it wus not significantly dillerent in the Cx45 were an inereusod duration of the P winve.and a Cx40 mice.Inerestingly.in the Cx prolonged and fractionated ORS complex in the ECGis in mice,the CV in the RHB wns significantly reduced hy 46% vivo Epicardial mapping indicated a changed and prolonged to 19.2+29 ms.The CVs in the 1.BB were not signifi- activatice of'the RV that ooukd be mttributed to slow condue- eantly diferent between the three genotypes investigated tion in the RBB.The CVs in ventricular working myocardi (Tablc). um,and the conduction delay in the AV node,were unaf- fected The durarion of the P wave was increased by about Discussion 18%nthex4 mice No decrease of the CV in the The targeted replacement of on Cx gere by another Cx gene RA was fourd in coctrast to the LA in which a significant has been performed for several Cxs Analysis of the reduction of about 23%was observed These data may resulting transgenic mee has indcaod th Cxs could lrve explain the increase of the P.wave duration although a either unique or rednckant fiunctiores,and has provikd dclayed aclivation betwoen the right and left atrium camot be impurtant clues regarding the role of Cxs in vivo.In this excluded,and indicate that Cx45 cannot fully replace the study,we have generated Cx4oKICxl5 mice,and assessed furction of Cx40.at last in the LA.Studies on Cxlo KO

recorded in a one-electrode wide strand, whereas LBB activation was electrically much wider and was always recorded in several electrode strands parallel to each other. In the Cx40/ mice, the CV in the RBB was 36.55.4 cm/s (Table), and it was not significantly different in the Cx40/KICx45 mice. Interestingly, in the Cx40KICx45/KICx45 mice, the CV in the RBB was significantly reduced by 46% to 19.22.9 ms. The CVs in the LBB were not significantly different between the three genotypes investigated (Table). Discussion The targeted replacement of one Cx gene by another Cx gene has been performed for several Cxs.36–38 Analysis of the resulting transgenic mice has indicated that Cxs could have either unique or redundant functions, and has provided important clues regarding the role of Cxs in vivo. In this study, we have generated Cx40KICx45 mice, and assessed the involvement of Cx45 in the propagation of cardiac impulse in the absence of Cx40. For both genetic backgrounds investigated, the major phenotypic effects of the biallelic replacement of Cx40 by Cx45 were an increased duration of the P wave, and a prolonged and fractionated QRS complex in the ECGs in vivo. Epicardial mapping indicated a changed and prolonged activation of the RV that could be attributed to slow conduction in the RBB. The CVs in ventricular working myocardium, and the conduction delay in the AV node, were unaffected. The duration of the P wave was increased by about 18% in the Cx40KICx45/KICx45 mice. No decrease of the CV in the RA was found in contrast to the LA in which a significant reduction of about 23% was observed. These data may explain the increase of the P-wave duration, although a delayed activation between the right and left atrium cannot be excluded, and indicate that Cx45 cannot fully replace the function of Cx40, at least in the LA. Studies on Cx40 KO Electrophysiological Parameters of Wild-Type and Mutant Mice Cx40/ Cx40/KICx45 Cx40KICx45/KICx45 ANOVA ECG parameters, ms (129Sv/CD1) RR interval 126.511 (n6) 122.58.1 (n11) 1205.6 (n10) 0.87 P wave 9.10.33 (n6) 9.30.4 (n11) 10.70.5 (n10)*† 0.034 PQ interval 34.80.7 (n6) 34.80.7 (n11) 35.81.1 (n10) 0.63 QRS duration 10.80.4 (n6) 10.50.3 (n11) 140.6 (n10)*† 0.001 ECG parameters, ms (129Sv/C57Bl/6) RR interval 104.35.6 (n14) 103.93.3 (n12) 1158.2 (n13) 0.36 P wave 9.40.31 (n14) 10.50.2 (n12) 12.20.5 (n13)*† 0.001 PQ interval 371.4 (n14) 36.40.9 (n12) 40.11.9 (n13) 0.19 QRS duration 10.20.2 (n14) 10.50.3 (n12) 12.50.5 (n13)*† 0.001 Ventricular activation (129Sv/CD1) Breakthrough RV (LV) 33% (33%) 11% (25%) 0% (0%) Apical/lateral RV (LV) 50% (50%) 89% (37.5%) 64% (92%) Basal/lateral RV (LV) 17% (17%) 0% (37.5%) 36% (8%) RV activation time, ms 5.40.4 (n6) 6.40.8 (n9) 90.7 (n11)*† 0.04 LV activation time, ms 6.10.8 (n6) 5.70.7 (n8) 6.70.5 (n12) 0.51 Conduction velocities, cm/s (129Sv/CD1) RA CV 30.81.8 (n6) 32.92.3 (n11) 30.02.3 (n12) 0.63 LA CV 31.51.6 (n6) 35.63.1 (n11) 24.32.2 (n11)*† 0.01 RV CV longitudinal 37.52.7 (n6) 33.16.7 (n10) 33.82.2 (n10) 0.45 RV CV transversal 21.03.4 (n6) 21.22.0 (n10) 19.60.6 (n10) 0.80 RV anisotropic ratio 2.00.3 1.70.2 1.70.1 0.52 LV CV longitudinal 46.34.2 (n6) 39.13.2 (n9) 38.83.2 (n12) 0.33 LV CV transversal 20.51.1 (n6) 18.61.1 (n9) 16.11.0 (n12) 0.11 LV anisotropic ratio 2.30.3 2.20.2 2.30.2 0.8 RBB CV 36.55.4 (n5) 44.65.6 (n9) 19.22.9 (n10)*† 0.001 LBB CV 47.87 (n5) 49.23.5 (n6) 485.8 (n6) 0.98 AV node function (129Sv/CD1) AV ERP, ms 68.34 (n6) 685.5 (n10) 74.25.1 (n12) 0.63 AV delay t, ms 55.72.5 (n6) 64.23.5 (n10) 64.92.4 (n12) 0.12 AV 31.55.7 (n6) 22.131.8 (n10) 24.42.4 (n12) 0.14 Values are given as meanSEM. *P0.05 compared to Cx40/; †P0.05 compared to Cx40/KICx45. n indicates number of independent experiments. Alcoléa et al Connexin40 Knock-In Connexin45 Mice 105

tions normally when Cx40 is replaced by Cx45. Numerous studies have described an increase in the PR interval and the Wenckebach cycle length in the Cx40 KO mice,18–21 which indicated an impairment of the AV conduction. Our results indicate that the absence of Cx40 in the AV node can be corrected by the expression of Cx45 despite differences in their intrinsic properties. The delayed activation of the RV, but not that of the LV, during SR, which increases the activation time of the heart, accounts for the increase of the duration of the QRS complex. Figure 6. Representative paced activation maps of the RA and LA (A and B), the RV and LV (C and D), and the RBB and LBB (E and F) of Cx40/, Cx40/KICx45, and Cx40KICx45/KICx45 mice. Position of the electrode array on the epicardium (A through D) or the septal myocardium (E and F) is indicated in the pictograms. Activation times are relative to the pacing stimulus in the maps of the RA, LA, RV, and LV and relative to the atrial activation in the maps of the RBB and LBB. CVs were calculated from these maps. Activation times (ms) are indicated by color codes. In the examples shown in A and B, the CVs in the RA and the LA were 36 and 29, 32 and 32, and 41 and 19 cm/s for Cx40/, Cx40/KICx45, and Cx40KICx45/KICx45 mice, respectively. In the examples shown in C and D, the CVs in the RV and the LV were 40 and 38, 40 and 33, and 30 and 34 cm/s for Cx40/, Cx40/KICx45, and Cx40KICx45/KICx45 mice, respectively. Note that the anisotropy of the conduction in the ventricles is independent of the genotype. CVs determined from the activation maps of the RBB and LBB illustrated in E (RS, right side of the septum) and F (LS, left side of the septum) were 42 and 47, 54 and 51, and 17 and 54 cm/s for Cx40/, Cx40/KICx45, and Cx40KICx45/KICx45 mice, respectively. Note that the activation patterns in the right and left BB are asymmetrical. Electrograms recorded in 2 different sites of both bundle branches are shown for each genotype. Alcoléa et al Connexin40 Knock-In Connexin45 Mice 107

106 Circulation Research January 9/23,2004 The CV in the vengneular workine myoeardium ws unat- References focted by the neplcemenl of Cx40 by Cx45.beer Ca40 is noc expressed in the ventricular working myocardium of the conduction and amtynogeress Covoase Pao 2001:10:169-177. adult mouse heart.The slow CV measured in the RBB of Cx40s mice.but not in the LBB.explains the 年.Q&rat12001,3435-472. abnermal activation of the RV,in these animals.In the nw 2:14:11-13 absenee of Cx41 Cx45 is the only known Cx which is expressed in the pruximal purts of the bunle branches 3 Dcutsch U,Sshl G.Stnclatal and funedorl divetsty ef ooenesin teres ndL2en.2238725-737 Assuming that Cx45 was upregulaed in a similar wy in hoth 5.Shrurol 1.Dupays 1.Theveriaa-Huaoy M,Alcrlea 5,Bry-aucterd bundle brmnches,its expression supports a nommal conduction in the LBB.but fails to do so in the RBB.indicating that ardee cerd由on wden.白Oaw人ed.elogww af she Can electrical ard/or structural differences between LBB and 1K:Wc30035-9w RBB nccount for the observed effeet.Aralysis of'mice in 6.Delame B.DaN E.Bamy Guicherd T.Manos I.Briad IP.llecke K. which oe allee of the Cx40 gene was replucd by FP GNe D ThevcrEau-louno M.Devdacmrcrtal bon ulsercuin 4u (Cx40 mice)has shown that the LBB was in fact a起SWo3 n moue hee1e r wit te diffeseslation of he aedichign写yrm.几tm1945:20:得-1. constituted with 20 or so parnllel strands coming from the His T.It.E brry-Gichnd T.lrand I-P.W'illacke K.Gms n. bundle,and covering entirely the left side of the imerventric- Thmmbu-Riiory M.3p四 ular septumn.In contrast,on the right flank the beanch (RBB) 道y Cevelepererlal stage当起%as,uaE1C1的 emerging fromn the His bundle was constituted by only one 15978423-437. .Ceppen sit.Dupe上 thin fiber in is pruximal pet Thus,the LBB is more extended and much more complex than the RBB,and this 1a上2到C年2g.1SK224 morphology is in agreement with the activation parterns 9.Coppen SR.Severs NJ,Gourdie RG. which were recorded (Figures 6E and 6F).Consequently. detnevtes an emended cenduction sysieme in the embnnic and monune ocerl hean.Der.199)2482-0 variations of the gap jutionl coupling in the ventncular CS 10.Vethijek EE vin Kenpen XU,Veerehild M Lurvink J,Jorgpata Hl, wil alTodt RBB conduction more severely than LBB cunduo- tion.Furthermure.te relaliorship betwcen the gap junctional n tebtion in cnmecn dianhnion rwdou M010-9 coupling and the CV in a strand of model cells is not linear. Stcnbers TH,Sm业E,Yaa山XAdo2 baboon o c2wi4ga At high levels of junctiocal conductance the CV saturates of comex43-defcien hears.Pes.200253: and significant reductions of the cocductance from very high 921-935. values do not necessanly result in a significant reduction of 12.Alcolea &Theveain-Riey M Jarry-Ciuichand T.Naries l, the CV.In contras.small roductions of'the conductnee fiom EChann JP,ISrand Jl',Mooman AF.Lmmr WH Ches II.Drwn lw values will kad lo draedic roductins ol'the CV.If onc 0 ert Cve Re人.199984136&-1379. assumes that the coupling is higher in the 1BB than in the 13.Coppen SR.Kodana I.Boyett MR.I.Y. RBB,which is not unlkely because the CV is higher in the H,Ych Hl,Severs N Coenesin5.mrajer onexn o the rhbil xirceltral so止，tae3-cprexc wilh cumnexnd3nafe山icisd at速hg LBB(47.8cms)ha▣in the RBB(65cms以a simil国 33et山Emlb3白.JMnscheat Cyocten9月4.90川s reduction of the intercellular ccupling in both bundle 14.Verherde S.van Kempen MI.Premra S.Rogk Mn.Jongsma 111 Gop brandes would te more prominem in the RBB than mn the LBB.The analysis of Cx4OKO mice cunfirmod that the RBB m。/内2 15.Kruge:O.Plun A.Kim L5,Winerhs E Maveier 5,Halln G. is much more sersitive to charges in the Cx expression than KircthofT S.Tr 0.Lemers WH Willcke K.DeSoctive veselr the 1BB.Indeed,the deletion of Cx40 without replacement develpnan in crnesin 45-daficiert mce.[hewipewt 2000127 resulted in conduction block in the RBB.but induced only a 410-4191 16.Kumo M NshiiK.Nikarun K.Tekeda N.Suaki M.Shben Y.Loss slowing down of the conduction in the LBB.The higher or cerneonk5 couses a custion defeat in carly cardiopenesis.Dverel wuinaabilly of thc RBB also rellocted m the cliical data wwl2000,2T350-3512 that indicale that the liogucncy of conduction impaittent in I,《G山in UE,MrGt,Tigraukkie H,V=l以5krMl山hm patients with cardiovascular diseases is much higher in the RBB than in the LBB (see for example Newhy et nl=) rttm好63 i mice wth民Tc1时0ofc0 In summary,the replacement of Cxlo by Cx45 resulted in 15 Siron AM.Goodenouch DA.Paul TX.Mice laeting comecin2 hwe the heart in a sigrificant reduction of the CV in the LA and a normd CV in the RA.The oundudion dcly in th AV nk nd bundl beagh bock.Cirr Aw.15988295-298 was unaflicled,wherets a partial loss of function became ducad candac cic1h1n的a11www道1nk3T1n题n apparent in the RBB,but not in the L.BB coanesin40-deficiert rice.192-2 20.HagendorT A.Schunacher B.Kirchhoft S.Luderiz B.Willecke K. Acknowledgments Condction cisourbances end inceosod omal vuinerabliy in cornesin40 leficient mke andlyzed by tasesoph超alU最Cireslaon Th work was supported by the CNRS,the Laiversite de la Mednerranee,e European Communay (oomtrt QLGl-1999. 2,5 CA.s,W'llodke K 00516).The Nether binds lleart Foundation (gramt 99-200).ad the French Mnistry of Fdueatinn (ACI Rinlogie de Developpemert rt 4了 4d.19910 Peysologe Integrative)S A was sipporied (fiellwships)by the 3n-139 Association Fratcabse corte les Myopethies,and the Guoupe de 22.Turroddas HS.Vaidya D.Smon AM.Pou DL Jalif:)Meky (iE. Reflexion sur Recherche Cardiovclare. Highodlutian optical muppre o te nghe bend e henng in coneesis40

The CV in the ventricular working myocardium was unaffected by the replacement of Cx40 by Cx45, because Cx40 is not expressed in the ventricular working myocardium of the adult mouse heart. The slow CV measured in the RBB of Cx40KICx45/KICx45 mice, but not in the LBB, explains the abnormal activation of the RV, in these animals. In the absence of Cx40, Cx45 is the only known Cx which is expressed in the proximal parts of the bundle branches.5 Assuming that Cx45 was upregulated in a similar way in both bundle branches, its expression supports a normal conduction in the LBB, but fails to do so in the RBB, indicating that electrical and/or structural differences between LBB and RBB account for the observed effect. Analysis of mice in which one allele of the Cx40 gene was replaced by eGFP (Cx40/KIeGFP mice) has shown that the LBB was in fact constituted with 20 or so parallel strands coming from the His bundle, and covering entirely the left side of the interventricular septum. In contrast, on the right flank the branch (RBB) emerging from the His bundle was constituted by only one thin fiber in its proximal part.40 Thus, the LBB is more extended and much more complex than the RBB, and this morphology is in agreement with the activation patterns which were recorded (Figures 6E and 6F). Consequently, variations of the gap junctional coupling in the ventricular CS will affect RBB conduction more severely than LBB conduction. Furthermore, the relationship between the gap junctional coupling and the CV in a strand of model cells is not linear.41 At high levels of junctional conductance the CV saturates, and significant reductions of the conductance from very high values do not necessarily result in a significant reduction of the CV. In contrast, small reductions of the conductance from low values will lead to drastic reductions of the CV. If one assumes that the coupling is higher in the LBB than in the RBB, which is not unlikely because the CV is higher in the LBB (47.8 cm/s) than in the RBB (36.5 cm/s), a similar reduction of the intercellular coupling in both bundle branches would be more prominent in the RBB than in the LBB. The analysis of Cx40KO mice confirmed that the RBB is much more sensitive to changes in the Cx expression than the LBB. Indeed, the deletion of Cx40 without replacement resulted in conduction block in the RBB, but induced only a slowing down of the conduction in the LBB.25 The higher vulnerability of the RBB is also reflected in the clinical data that indicate that the frequency of conduction impairment in patients with cardiovascular diseases is much higher in the RBB than in the LBB (see for example Newby et al42). In summary, the replacement of Cx40 by Cx45 resulted in the heart in a significant reduction of the CV in the LA and a normal CV in the RA. The conduction delay in the AV node was unaffected, whereas a partial loss of function became apparent in the RBB, but not in the LBB. Acknowledgments This work was supported by the CNRS, the Université de la Méditerranée, the European Community (contract QLG1-1999- 00516), The Netherlands Heart Foundation (grant 99-200), and the French Ministry of Education (ACI Biologie du Développement et Physiologie Intégrative). S.A. was supported (fellowships) by the Association Française contre les Myopathies, and the Groupe de Réflexion sur la Recherche Cardiovasculaire. References 1. Kanno S, Saffitz JE. The role of myocardial gap junctions in electrical conduction and arrhythmogenesis. Cardiovasc Pathol. 2001;10:169–177. 2. Harris AL. Emerging issues of connexin channels: biophysics fills the gap. Q Rev Biophys. 2001;34:325–472. 3. Evans WH, Martin PE. Gap junctions: structure and function. Mol Membr Biol. 2002;19:121–136. 4. 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浙江大学文本 Replacement of Connexin40 by Connexin45 in the Mouse（cbp）_生理学

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