nshsooy aJla A903 USA. 92093, CA Jolla, La Anesthesiology, tblzn onxn4-otiiggpjntosi cardiac for in myocytes essential junctions is gap and connexin-43-containing occludens-1 stabilizing zonula binds directly Vinculin ARTICLE RESEARCH ß 1104 2013 December 4 Accepted 2013; October 1 Received and 2 and (Gilmore 2004) al., 1 phospholipids et (Izard talin acidic 1996), al., as et When Weekes such 1996; junctions. Burridge, ligands, cell–cell and to cell–matrix bound both in found protein result, 2010). might al., al., et et instability (Kostin Tirziu failure heart membrane 2000; and remodeling impaired, pathological to is leading maintained. adequately anchorage myocytes, be cells cardiac If other between and anchorage cardiac matrix strong extracellular relaxation, a the that Thus, and essential stress. is mechanical contraction it significant under of normally are cycles myocytes continual With INTRODUCTION Zonula Heart Vcl, ZO-1, Vinculin, occludens-1, junction, Gap myocyte, junctions Cardiac and WORDS: gap KEY ZO-1 stabilizing in and role Vcl crucial integrity. myocyte a between and plays interaction first Vcl the how direct is illustrates a this summary, and showing In Erk1 necrosis. study Akt, and cardiomyocyte of (4) activation talin, and increased Erk2, ZO-1, (3) activation, Cx43, decreased PI3K Vcl (1) reduced of to of leading expression amount expression the protein protein co- in and reduction transcript Vcl the a mRNA in dics. caused of ZO-1 cardiomyocytes Excision intercalated bound mouse studies. directly from and two-hybrid ZO-1 ZO-1, yeast and of this Cx43 loss of immunoprecipitated Vcl loss parallel cardiomyocyte of that Loss and caused disc. Cx43 intercalated and the Vcl, at that interact colocalized found ZO-1 We ZO-1 junctions. gap we and destabilizes death, interaction Vcl sudden and that arrhythmia to hypothesized function heart preserved sarcolemma. maldistribution mice with Vcl-knockout causes the cardiomyocyte-specific cardiomyocytes predisposes to and from Cx43 cytoskeleton Vcl of of actin loss the that anchor Given ZO-1 actin. and and cadherin GJA1), Vcl TJP1) as known as also known (Cx43, cadherin-based also connexin-43 and binds (ZO-1, integrin- occludens-1 Zonula to junctions. filaments cellular actin links (Vcl) Vinculin ABSTRACT Zemljic-Harpf E. Alice Ato o orsodne([email protected]) correspondence for *Author 92093, CA Jolla, La Cardiology, Medicine, of USA. Department Medicine, of School nraA Domenighetti A. Andrea nvriyo aiona a ig colo eiie eatetof Department Medicine, of USA. School 92161, Diego CA San Diego, California, San of Diego, University San Healthcare Administration Veterans 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,10–16doi:10.1242/jcs.143743 1104–1116 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. icln(c)i natnbnigadmembrane-associated and actin-binding an is (Vcl) Vinculin 1,2 oehC Godoy C. Joseph , 3 n oetS Ross S. Robert and 3 nvriyo aiona a Diego San California, of University Vcl eei neonatal in gene b Ditgi,(2) 1D-integrin, 1,3 lkad Platoshyn Oleksandr , 1,3, * einadfeil al(ogne l,2004). al., 2007). et hinge (Borgon head, al., globular tail a flexible full-length et of and of assembly Hu region the structure described crystal cadherin/catenin-containing 2010; and the Vcl, integrin- human al., determined or has et either group actin Izard’s (Grashoff through the adhesions junctions assemble membrane to adherens focal cell and of anchor the One containing to 2006). to is al., Vcl cytoskeleton et of (Ziegler features activation unique Vcl initiating thereby a ssga rndcin(iPooadD ail,20) The (4,5)-bisphosphate 2006). Camilli, phosphatidylinositol De are and [PtdIns(4,5) They phospholipid Paolo 1993). membranes. (Di acidic transduction cell Gimona, well signal as eukaryotic interface, and as membrane–cytosol the of organizing Niggli for at side important 1986; positioned cytosolic phospholipids al., the integral are et Phosphatidylinositides (Niggli bilayer amre l,20) ls hshioiie3knss(PI3Ks) 3-kinases phosphoinositide I PIP Class including phospholipids 2009). 2005; al., phosphorylate al., et (Chandrasekar et cytoskeleton coupling actin the Palmer the regulate to phospholipids sites adhesion how of modify can that sensor ncricmoyecl uvvl(hg ta. 02.PIP 2012). al., functions et key (Ghigo including survival cell cells, myocyte many cardiac in in roles numerous plays which rtisbn ag ubro atesicuigcnei (Cx) connexin including partners and of proteins number ZO large 1994). a Goodenough, and bind Jesaitis proteins gap 2003; as al., well et as junctions, (Inoko adherens junctions and They junctions respectively). tight TJP3, in and present TJP2 are TPJ1, as PI3K known (also affect proteins might to expression talin, unclear. protein and currently Vcl is actin activation altered 1996). as Burridge, how and such However, (Gilmore proteins, sites adhesion adhesion cell integrin-containing focal recruits Vcl YnadJne,20) c id PIP binds Vcl 2003). proteins Janmey, regulatory actin and of targeting (Yin and activity the modulating dpes(ot n ish 00.Hne IK a generate can PI3Ks Hence, 2010). (3,4,5)-t Hirsch, and phosphatidylinositol (Costa adapters yE15(asn ta. 2008). al., et (Katsuno E8.5 E11.5 lethality by from embryonic to leads development This angiogenesis. the and abnormal evident, including in defects extra-embryonic growth gene and embryonic ZO-1 both delayed with the onward, 1998; in of Moolenaar, deletion results and Global PDZ mouse 1998). (Giepmans second al., GJA1) et protein the as junctional Toyofuku myocytes, gap known the (also cardiac with 2009; interacts Cx43 In directly Anderson, ZO-1 2002). of and domain al., important (Fanning et as proteins Fanning role their scaffolding highlighting cytosolic afadin, and F-actin nephrin, atnn(ose l,20) nrmlclrbnsaeunmasked, are bonds intramolecular 2006), al., et (Bois -actinin c skont netit h acidic-phospholipid-containing the into insert to known is Vcl h ouaocues(O aiycnit fZ-,- n -3 and -2 ZO-1, of consists family (ZO) occludens zonula The P 2 lzbt .Asfaw K. Elizabeth , 2, a -actinin, PIP 2 ietyrgltsteatnctseeo by cytoskeleton actin the regulates directly ] a ctnn cldn lui,shroom-2, claudin, occludin, -catenin, ihsht [PtdIns(3,4,5) riphosphate 1,3 naR Busija R. Anna , 2 n c sscaffolding as act and , 2 hrb ciga a as acting thereby , P 2 -activated 3 ,PIP 2 , 3 ],

Journal of Cell Science EERHARTICLE RESEARCH eesr o O1pstoigi h necltdds.Heart disc. intercalated the in positioning ZO-1 for 1B, necessary (Fig. next tissue cardiac we normal tissue. in was partner, colocalization there asterisks). cardiac interacting ZO-1 that in and suggested colocalized Cx43 sections Vcl ZO-1 transverse known Given of and Immunostaining a Vcl asterisks). whether 1A, is (Fig. assessed the ZO-1 at disc was localization ZO-1 intercalated that to addition corresponding myocyte pattern In a cells, cardiac transverse 1A). a endothelial (Fig. had in myocytes capillary seen ZO-1 cardiac Given in to PECAM. pattern adjacent tissue. expression marker cardiac performed and longitudinal ZO-1 endothelial-cell-specific we normal both endothelium, for the antibodies in capillary with in staining ZO-1 immunofluorescence expression levels Hence, known assessed Cx43 2004). its in al., first alterations et to Zemljic-Harpf we linked 2007; are al., levels tissue et decreased (Zemljic-Harpf cardiac that in demonstrated disc Vcl have intercalated of we the studies, to localization previous ZO-1 In for necessary is Vcl RESULTS fti neato ol etblz x3cnann gap Cx43-containing abnormalities destabilize cause integrity. and myocyte function would in loss junction that gap interaction and perturb mouse interact, junctions, the ZO-1 this in and 2007; Vcl findings of that al., these hypothesized Given et we 2004). (Zemljic-Harpf heart, al., normal et was Zemljic-Harpf function heart mouse g ZmjcHrfe l,20) motnl,abnormal Importantly, within Vcl detected 2007). both was of Cx43 discs al., protein intercalated the junctional of gap et months of 6 distribution by (Zemljic-Harpf failure heart developed of age period, died this and suddenly past cardiomyopathy survived cardiac dilated died systolic that preserved mice and despite cVclKO life, of we function. tachycardia weeks 10 ventricular first the cardiac- addition subset within displayed of a phenotypes; them means In distinct by two of developed heart pressure- mice Vcl 2004). the mouse cVclKO These the to in normal of al., Vcl studied inactivation adaptation of that myocyte-specific role et previously cardiac showed the investigated for We (Zemljic-Harpf and essential 1998). mice overload is Vcl al., expression null et hemizygous abnormal (Xu including defects multiple cardiogenesis have mouse embryos the Vcl-null of E10. cardiac deletion of Global alignment 1997). and Vcl al., assembly et ordered of (Shiraishi as determination intercalated myofibrils the well and for as important costameres shape be to cell at shown been expressed has and highly discs is It 2011). myocyte. al., et plaques Rhett junctional 2012; gap Gourdie, the and into of (Rhett incorporation abundance for the the as regulates available referred and Cx43 also ZO-1 in plaque, with (Hunter found junctional colocalizes Cx43 gap perinexus, distribution that a suggested surrounding and have area peripheries, reports the size Previous junction 2005). junction gap al., at et gap accretion ZO-1 regulating Cx43 that proposed of thereby been (Decrock has rate It death the 2007). al., cell controls et Kardami and 2009; al., regulation et as growth such differentiation, processes cell physiological active further coordinating biologically They small thereby 2000). and molecules, al., ions et of Saffitz transport and 2011; intercellular permit al., (Delmar et myocytes Rhett cardiac 2012; Liang, couple electrically therefore and et easse hte rprVlepeso was expression Vcl proper whether assessed we Next, c a enlne oipratfntoswti h cardiac the within functions important to linked been has Vcl potentials action of conductance rapid for allow junctions Gap eecue mroi rwhrtrainwt ehlt by lethality with retardation growth embryonic causes gene +/ 2 n VlOmc hnthe when mice cVclKO and Vcl ee(cVclKO). gene ooaie ihVl n htnra c xrsinis expression it Vcl the at cardiomyocyte-to- normal ZO-1 where of that positioning at and disc. connections, and expression intercalated proper expressed Vcl, for disc necessary primarily with intercalated colocalizes is the cardiomyocyte ZO-1 sites indicate colocalization findings adhesion these of no cell together, that Taken Vcl-containing asterisk). but arrangement double lateral 2A, at asterisks), (Fig. ‘step-like’ identified 2A, be (Fig. the lateral could the 2A, discs to cardiomyocytes. (Fig. on intercalated detected discs owing adult was intercalated expression sarcolemma isolated at ZO-1 seen Patchy used asterisks). again was we also Colocalization disc, were double in 1C, intercalated and (Fig. detected bed, heart still the capillary in were within only asterisks). the non-myocytes ZO-1 Vcl in within and lacked visualized Vcl cells mice from cVclKO cells, endothelial absent that muscle was Given Vcl cardiac intercalated 1C). when the (Fig. at that detected longer disc found no was we ZO-1 myocytes, and cardiac used was (cVclKO) mice Vcl-knockout cardiac-myocyte-specific the from tissue yctsadwe c sdltdfo ada yctsZ- salso is ZO-1 myocytes cardiac from cardiac deleted wild-type in is lost. discs Vcl intercalated when at and colocalize ZO-1 myocytes and Vcl 1. Fig. O1i nw odrcl idt x3i ada myocytes expression Vcl cardiac that showing Given in data 1998). prior Cx43 our al., and et to above findings Toyofuku bind our 1998; directly Moolenaar, and to (Giepmans known is ZO-1 ZO-1 with interacts directly Vcl (*). colocalize Vcl and ZO-1 and disc, 20 intercalated bar: the Scale myocytes, at cardiac distinctly the is Within myocytes. ZO-1 cardiac cell to endothelial adjacent substantial expression cardiac the ZO-1 highlights between expression cells PECAM longitudinal endothelial This a marking myocytes. showed with staining consistent PECAM pattern anti- (B). staining or antibodies (A), anti-ZO-1 antibodies and anti-ZO1 Vcl and anti-PECAM using immunomicroscopy hr eetdi nohla el n o-yct elppltos(**). populations cell non-myocyte and cardiac-myocyte-specific Vcl cells Upon and endothelial ZO-1 in antibodies. detected anti-ZO-1 where and anti-Vcl using immunomicroscopy oesl iulzditraae ic hncmae ocontrols. to are compared there when Note discs parallel. in intercalated lost visualized was expression easily ZO-1 no and cell the from lost was ofrhrso htVladZ- ooaie tthe at colocalized ZO-1 and Vcl that show further To AB idtp os er isewseautdby evaluated was tissue heart mouse Wild-type (A,B) ora fCl cec 21)17 1411 doi:10.1242/jcs.143743 1104–1116 127, (2014) Science Cell of Journal m .()ccK er isewseautdwith evaluated was tissue heart cVclKO (C) m. Vcl eedlto ccK) c expression Vcl (cVclKO), deletion gene 1105

Journal of Cell Science EERHARTICLE RESEARCH 1106 5 the spanning and Vcl, sequences of prey full-length ZO-1 the designed spanning constructed We constructs ZO-1. also bait and Vcl Vcl of of series interaction a direct for test to screen cardiac and isolated and and heart Cx43 tissue 2B,C). whole (Fig. the Vcl, both myocytes cardiac that in complex showed a form with results ZO-1 performed Our we antibodies lysates. First, cardiomyocyte anti-Cx43-specific other. anti-ZO-1 each anti-Vcl, and using to studies and muscle directly immunoprecipitation ZO-1 cardiac reciprocal whether bind the investigated proteins in we Vcl 2007), placement al., Cx43 et proper (Zemljic-Harpf for necessary is a as used was IgG subjected antibody. were anti-Vcl control. a samples an negative IP formed using ZO-1 Vcl immunoblotting and that IP Vcl show Cx43 to ZO-1. to and used Cx43 Wild- further with (C) were complex respectively. lysates immunoblot antibodies, heart to anti-Vcl whole subjected or type were anti-ZO-1 samples either IP with ZO-1 analyses antibodies. and anti-Cx43 Vcl-IP co- or the were anti-ZO-1 Then (CMC) anti-Vcl, myocytes with mouse cardiac (IP) wild-type isolated immunoprecipitated from or lysates (Heart) Protein heart border myocytes. whole cell cardiac lateral isolated the at and not heart but 20 (*) bars: disc Vcl Scale intercalated of the (**). colocalization at showed yellow myocytes in cardiac ZO-1 of and wall cell intercalated lateral the the of and image disc disc enlarged non-intercalated the the panel: Lower along membrane. found intercalated sarcolemmal the was of colocalization ZO-1 arrangement No of ‘step-like’ staggered disc. expression known lateral intercalated the apparent the to The owing at was myocytes. colocalize cardiac ZO-1 wild-type and of Vcl cardiac disc panel: in Upper complex myocytes. scaffolding cardiac a form ZO-1 and myocytes. Cx43 Vcl, 2. Fig. h ullnt O1tasrp Fg 3A). (Fig. transcript ZO-1 full-length the o idayitrcinbtenVlfamnsadteC- the kb and 2.5–5.2 the fragments by for Vcl coded region between did (the we ZO-1 interaction note, of domain Of any 3B). terminal (Fig. find ZO-1 of both not 2.5kb domain first Vcl the N-terminal with the interacted and Vcl full-length that revealed et epromdayattohbi YH interaction (Y2H) two-hybrid yeast a performed we Next, A c n O1clclz nitraae ic fisolated of discs intercalated in colocalize ZO-1 and Vcl (A) m .()VladZ- oimnpeiiaei whole in co-immunoprecipitate ZO-1 and Vcl (B) m. b glcoiaeassays -galactosidase 9 . bof kb 2.5 rncitepeso ob eue o51 to and protein reduced Vcl be caused to excision expression gene transcript Vcl Cre-mediated point rmAVCe rAVLc-netdVcl AdV-LacZ-infected or samples AdV-Cre- on performed from were studies RT-PCR and Immunoblotting ausadnraie oGPH(i.5) h eesof levels The 5A). (Fig. GAPDH to normalized and values (21 ZO-1 (21 including: significantly proteins, found Vcl-associated (61 we of reduction, expression protein reduced Vcl With 5A,B). (Fig. 27 at to attached cells well used remained we matrix-coated cells and matrix, control similar and from morphologically Vcl-deficient were (data analyze detached that reduced to profoundly Therefore, longer became shown). levels not or protein Vcl the h as 72 plates, for maintained nta tde,w on htCeifce Vcl Cre-infected that found we studies, initial os etiua ycts(MM)ioae rmVcl from isolated (NMVMs) (Vcl ‘floxed’ myocytes neonatal homozygous using in ventricular system expression culture Cx43 cell mouse a control to might turned Vcl we cardiomyocytes how elucidate further Cx43 To as of proteins that Vcl-associated as of well expression protein reduces ablation Vcl ol fettemmrn oaiaino x3 eepce a expected We Cx43. of at reduction localization expressed Vcl membrane whether both experimental the test were affect to Vcl our would wanted and we cardiac- For because Cx43 sarcolemma that antibody. the the crucial was anti-Cx43 with it an design, immunolabeled with anti-sarcomeric- colabeled were in evident were cells NMVMs myocyte-specific pure cultures, ensure To our deleted. be could gene MM eie rmVcl from derived NMVMs study. culture this confluent of utilized remainder we the findings, for these conditions on Based in 4C). to monolayer (Fig. they akin more intact the densities, along membrane, an detected cell were contacts higher contacts formed cell-to-cell cell–cell Cx43-containing at NMVMs at In which seen Confluent was grown 4A). Cx43 4B). Cx43 (Fig. (Fig. and were perinuclear sites, localization contact culture cells formed intense membrane sparse when in sparse displayed contrast, grown and only were expression NMVMs they when conditions, that found NMVMs in but pattern staining Cx43 membrane-localized prominent xrsino opooywsdtce nteeAdV-Cre- these in NMVMs detected protein Vcl WT was Vcl floxed in morphology infected change (no No or NMVMs AdV-Cre. to (WT) expression them wild-type exposed and used treatment allele) also AdV-Cre further to we owing To effects 2003). alone, side al., potential expressing any et for (Iwatate AdV control controls as recombinant served a (AdV-LacZ) with Cells (AdV-Cre). infected Cre-recombinase expressing (AdV) adenovirus eunea at gis ullnt c spe Fg 3C). (Fig. prey as coding Y2H Vcl minimal ZO-1 full-length the additional a against of performed fragments identify bait, smaller as we To of sequence series shown). ZO-1, a using not in analysis (data domain ZO-1) interacting of portion hs idnsidct htVldrcl id oZ- nadult with in complex ZO-1 scaffolding Cx43. to a and binds forms SH3 ZO-1 thereby directly plus and together, Vcl myocytes, PDZ Taken fragments that cardiac 3D). third indicate (Fig. only the Vcl findings with these or interacted that domain, ZO-1, of PDZ revealed domains third the assays containing activity galactosidase ordc c xrsini hsmdlsse,w infected we system, model this in expression Vcl reduce To 6 6 6 6 %( 2% 3%, 1%, 2%, ora fCl cec 21)17 1411 doi:10.1242/jcs.143743 1104–1116 127, (2014) Science Cell of Journal P P P P , , , , .01,o oto netdcl aus respectively values, cell infected control of 0.0001), .01,C4 (17 Cx43 0.0001), .0) ausbigepesdrltv ocontrol to relative expressed being values 0.001), .01,tln(43 talin 0.0001), , 0hps-neto.A hs6- time 60-h this At post-infection. h 60 ( fl/fl upeetr aeilFg S1 Fig. material supplementary flox/flox nvivo in os ers nwihthe which in hearts, mouse ) 6 6 8%, 6%, a iewt recombinant a with mice odtosi ada tissue cardiac in conditions atnnatbd,and antibody, -actinin P P , , 0.0001), .01,adtubulin and 0.0001), 6 flox/flox %( 4% b -galactosidase P b flox/flox MM.In NMVMs. , Dintegrin 1D .1 and 0.01) cells Vcl b ) - .

Journal of Cell Science EERHARTICLE RESEARCH neto.Uo h euto fVlpoen membrane assess protein, specifically To Vcl 6A). (Fig. of lost was reduction at Cx43 analyzed the of were localization Upon cells cell this above, infection. in As performed model. next communication culture junctional was gap analysis in decrease Immunohistochemical a protein perturbed. causes cell, also reduction the is Vcl proteins from Vcl-related lost in of is function range Vcl a and as of expression that, expression protein but Cx43 myocytes, that only indicate cardiac data not These controls 5B). 7) (Fig. Vcl and controls significantly (3 to not domains compared were SH3 changed proteins plus Vcl-associated PDZ these third the of or transcripts 6) were and ZO-1 (2 p53-1. for PDZ and sequences third a-TD1 coding the were c partial containing controls and Her from ZO-1 negative c-3 experiments. produced of and (assays two-hybrid Constructs fragments p53-TD1 ZO-1 yeast ‘a’. Only were with of construct (D) controls interact set as a-2 2–7. Positive not second designated and fragments Vcl. did a and a-3 as domain with in prey (assays designated Vcl interacted interactions based as ZO-1 and C-terminal Vcl–ZO-1 Constructs used with bait the the c. was interacted as contrast refine (B) and construct Vcl used In 3. to b kb) full-length and b-2). used a, (0–3.2 2 that and constructs as 1, Vcl show b-3 additional labeled as length results (assays labeled of are Graphical and ZO-1 representation These ZO-1. prey k with Schematic assays. as with (0–3.2 interacted (C) two-hybrid used Vcl directly also were yeast full-length interacted domain kb) in for Vcl Vcl 1.5–2.5 bait sequences of and N-terminal coding as kb region on used 0–1.5 N-terminal based and kb, the made kb), (0–0.25 were that ZO-1 1.6–3.2 Constructs for and interaction. sequences kb protein coding for (0–2.5 ZO-1. on assay Vcl with to of interacts used mutants directly fragments deletion Vcl ZO-1 that and white) showing tail, screening and two-hybrid Yeast 3. Fig. , 0hpost- h 60 a ucin Fg B.We epromdsimultaneous into performed Yellow Lucifer we (Vcl and cells control and dextran When NMVMs, through Rhodamine junctions, directly 6B). of pass (Fig. gap microinjection to large through junctions too easily 2000) gap is Rivedal, which transported Yellow, and dextran, Lucifer be dyes: Rhodamine Opsahl two 2011; used can we al., studies which these et el- For 2009; . Li 6B,C) al., microinjection 1987; et (Fig. (Boswell al., dye assays et loading performed scrape Fouly as then well as between we studies communication NMVMs, intracellular and connecting function junction gap uie elwdetase oajcn MM,btn transfer no but NMVMs, adjacent to transfer dye Yellow Lucifer A ceai ersnaino c ha,ga;hne black; hinge, gray; (head, Vcl of representation Schematic (A) ora fCl cec 21)17 1411 doi:10.1242/jcs.143743 1104–1116 127, (2014) Science Cell of Journal fl/fl b lsAVLc)soe extensive showed AdV-LacZ) plus glcoiaeatvt sasrevealed assays activity -galactosidase .The ). ,afull- a e, ) and b), 1107 -2).

Journal of Cell Science eue eeso IKpopoyae nY5 PI3K Y458 on cells, phosphorylated these PI3K in reduced of was Vcl levels When reduced 7A). (Fig. analyzed were elwtase oclsdsatfo h cac,indicating cells scratch, Vcl-deficient the whereas from function, distant (Vcl junction cells gap to appropriate transfer Yellow xrsini ada yctsi eesr o prpit gap appropriate together, for necessary Vcl function. Taken is proper junction myocytes that cardiac place. indicate in data expression took scrape-loading significant and communication no microinjection that indicating junctional scratch, the gap to adjacent cells to only EERHARTICLE RESEARCH 1108 (0.70 lysates Whole-cell Vcl AdV-LacZ-infected ERK1/2). and and AdV-Cre- from Akt derived (e.g. survival pathways downstream in signaling proteins and expression (PTEN) homolog key homeostasis. affected cardiomyocyte Vcl for myocyte necessary of and molecules loss Cx43 how signaling ZO-1, evaluated next of that we and expression protein, interacted, reduced Cx43 Vcl and ZO-1 of Vcl, loss that myocytes found cardiac we in that signaling Given PI3K/PTEN inhibits disruption Vinculin dyes dextran Rhodamine and (Vcl Yellow cells scrape Control Lucifer with 6C). system (Fig. the culture our of in loading to assay companion transfer a performed dye Yellow Lucifer 6B). (Vcl (Fig. little cells cells showed neighboring Vcl-deficient the AdV-Cre) whereas plus dextran, Rhodamine of 2 bars whole Scale the sited. 20 along adhesion expression cell-to-cell sites. Cx43 marking contact strong sarcolemma, cell–cell display at cultures only Confluent sarcolemmal- found (C) contact, was cell–cell expression sarcolemma. close Cx43 the in localized along were Cx43 cardiomyocytes of When Cx43 expression (B) perinuclear weak dominant only showed and cells expression plated Sparsely (A) anti-sarcomeric antibodies. sites. with junction colabeled cell–cell dense were of requires myocytes formation Cx43 allow myocyte to cardiac conditions of culture localization Membrane 4. Fig. oprdbtenLc n r ape,wr detected. were samples, pAkt Cre and phosphorylated LacZ Interestingly, between compared (0.42 o hs ecoefrtt sesP3,popaaeadtensin and phosphatase PI3K, assess to first chose we this, For also we communication, junction gap evaluate further To m (C,D). m fl/fl 6 6 0.03, 0.09, lsAVCe ipae uie elwdetransfer dye Yellow Lucifer displayed AdV-Cre) plus P P , , .5,PE (0.60 PTEN 0.05), .0) l odcag eaiet GAPDH, to relative fold-change all 0.005), fl/fl lsAVLc)soe Lucifer showed AdV-LacZ) plus 6 0.11, S473 a atnnadanti-Cx43 and -actinin P n pAkt and , .5 n oa Akt total and 0.05) b fl/fl Dintegrin 1D m T308 (A,B), m NMVMs Cardiac (Y458) were fl/fl ae ssonhr oalwdrc oprsnwt rncitexpression transcript with comparison samples. direct same allow to the here in from shown expression is protein A of panel analysis Densitometric cells. myocytes control cardiac LacZ-treated associated the talin, of ZO-1, levels proteins, transcript the alter significantly netdVcl infected nteVldfcetcls hsyedda nraei normalized in increase an yielded reduced, This Akt significantly cells. was Vcl-deficient Akt the in total as increased, independently ** rncitaayi. B R-C a sdt sa ape rmVcl from samples assay with to together used B was Panel qRT-PCR in (B) displayed are analysis.) all data transcript were these and proteins GAPDH individual to the normalized of analyses (Densitometric control. rncit a eue yCemdae eeecso (27 excision GAPDH gene to Cre-mediated ** normalized by expression, reduced transcript was At Vcl infection. transcript, AdV-Cre infection, via post above h as reduced 56 was Vcl which in cardiomyocytes { expression Vcll alters of proteins. myocytes infection cardiac associated Cre in of protein range Vcl a of of Reduction 5. Fig. hs aaidct htrdcino c nteNV ed to leads NMVM RhoA the in but Vcl of deficiency, reduction 7C,D). that Vcl (Fig. indicate reduced data by significantly These was affected expression protein not kinase were actin- dystrophin proteins and membrane- The 7A,B). cytoskeleton-associated (Fig. expression ERK1/2 (2.10 total (4.65 ERK1 activation in changes ERK2 fold induced also (5.94 c rti xrsint 51% to expression protein Vcl d-r netdclscmae ocnrlifce el a eue as: reduced was cells infected control (61 to ZO-1 compared cells infected Adv-Cre P P P , , , .0) AD rti xrsinwsucagdadue sloading as used and unchanged was expression protein GAPDH 0.001). T308 0.0001), .01.Hwvr c euto ncricmoye i not did myocytes cardiac in reduction Vcl However, 0.0001). 6 0.08, tk (4.00 /tAkt 6 ora fCl cec 21)17 1411 doi:10.1242/jcs.143743 1104–1116 127, (2014) Science Cell of Journal % ** 2%, fl/fl b adoycts ihti efudpoenepeso fthe of expression protein found we this With cardiomyocytes. Ditgi (21 integrin 1D P , P , fl/fl .01 esscnrlsmls c reduction Vcl samples. control versus 0.0001) .01,C4 (17 Cx43 0.0001), b VlO entlmuevnrclrmoye reduced myocytes ventricular mouse neonatal (VclKO) Ditgi,C4 rtblnwe oprdt AdV- to compared when tubulin or Cx43 integrin, 1D 6 0.32, 6 6 0.06, 6 ,* 4, % ** 1%, P , A muoltigrvae htAdV- that revealed Immunoblotting (A) P P .1 oprdt oto (AdV-LacZ) control to compared 0.01, P , 6 , , % ** 8%, .0)adAkt and 0.001) .0) hnnraie to normalized when 0.001), .01 n uui (21% tubulin and 0.0001) a -actinin, P , 6 .01,tln(43 talin 0.0001), 0.07, b P ctnnand -catenin 6 , %o control, of 2% .0)and 0.001) S473 6 6 3%, 6%, /tAkt fl/fl

Journal of Cell Science EERHARTICLE RESEARCH IKatvto ly nipratrl nmyocardial growth, cellular in for essential role is function important junction and contractility, gap an cardiac normal and plays signaling pro-survival metabolism, activation injury cellular PI3K rapid causes reduction Vcl ERK1/2. and activation Akt associated both with of PI3K/PTEN, through signaling altered Vcl in expression cell–cell and dysfunctional localization, causes protein and communication. Cx43 expression, disrupts Cx43 NMVMs and in ZO-1 reduction Vcl 6. Fig. a ucincmuiaini h c-eiin ada myocytes. cardiac Vcl-deficient defective the showing in cells, again communication control cultures, AdV-LacZ junction Vcl-deficient in gap AdV-Cre-treated place in took cell not only with but transfer consistent dye loading, Yellow scrape Lucifer dyes. cell injury. Yellow the Lucifer marked and dextran Scrape dextran (C) Rhodamine cells. Rhodamine Vcl-deficient using the assays in cardiomyocytes, loading function neighboring junction to gap spreading reduced (AdV-Cre) dye indicating cells Yellow Vcl-deficient Lucifer but no junctions, myocytes showed cardiac gap adjacent functional with to showed transfer consistent (AdV-LacZ) dye cells Yellow dextran Control Lucifer Rhodamine myocytes. extensive observed. cardiac was injected cells the evaluation neighboring labeled NMVMs, to of into transfer (L.Y.) microinjection dye Yellow simultaneous of Lucifer After and (B) (R.D.) cell–cell VclKO. dextran and in Rhodamine perinuclear found both was from AdV- sites Cx43 to contact of compared Loss expression cells. protein control Cx43 LacZ-infected of reduction visible significant fl/fl A muoirsoysosta euto fVcl of reduction that shows Immunomicroscopy (A) MM sn dnvrlCe(d-r)rslsin results (AdV-Cre) Cre adenoviral using NMVMs ees a eetdi h c-eiin el scmae to compared as cells LDH Vcl-deficient Increased the medium. in culture a into detected for cells was measured assess the we release to reduction, by protein release Therefore protein Vcl LDH of damaged. effect LC3 is cytotoxic potential membrane and plasma Vcl Beclin-1 the of reduced reduction with 7E,F). detected (Fig. parallel expression we In or 7E,F). 3 protein, (Fig. caspase apoptotic expression, cleaved inhibited and Vcl PARP either were markers reduced apoptotic the With of the of in pathways. expression activation death reduction cell Vcl triggered autophagic whether system evaluated NMVM combined next detached this and we Given shown). shrinkage information, not cell (data had plates NMVM matrix-coated from that found we protein system) Vcl of 2007). reduction al., a et of upon with (Zemljic-Harpf above, consistent discussed myocytes as cardiac hearts, Furthermore, of mouse loss and cVclKO death in fibrosis of replacement loss pathways. how from death evaluated cell junction next loss affects gap we Vcl Vcl Cx43, signaling, myocyte that of survival show expression and studies disturbed function al., in above et results our Kardami myocytes 2009; that al., Given et 2007). 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(Fig. controls paau fajcn ycts hrb teghnn the strengthening and thereby adherens myocytes, fascia adjacent contractile the of Both bridging apparatus linkages, desmoplakin, plakoglobin. mechanical via form termini desmosomes and muscle al., the et link plakophilin and to (Maiers been disc filaments junctions intercalated has the intermediate adherens at it localized to Recently are Desmosomes barrier 1998). 2013). endothelial al., the to et of to recruitment Weiss ZO-1 binding that 2010; for described al., known through et also sites (Peng is cytoskeleton contact and actin cell–cell 2005), the cadherin-based can to al., Vcl sites and et 1983). adhesion al., (Craig (Chandrasekar of et coupling Pardo known 2013; the well al., regulate et is Dumbauld maturation 1983; focal Pardo, in and involvement its formation and costameres adhesion at communication Vcl junctional of gap localization in The myocyte Vcl of and role Novel function junction intercalated gap the at normal complex survival. ensures protein that a that suggest form cardiac data disc ZO-1 Our increased and processes. 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Fig. ARTICLE RESEARCH c neat ihpopoiisi h lsammrn,by membrane, plasma PIP the in binding phospholipids directly with signaling interacts PI3K/Akt in Vcl Vcl of role Novel in role important an of plays ZO-1, that localization decreased linker membrane caused cytoskeletal protein actin Vcl crucial of and reduction that Cx43 and showed mild model adherens culture a myocyte between our even linker in that data molecular The propose a junctions. we gap as ZO-1, is binds function it Vcl might that that Vcl shows directly study Given junctional our with Vcl circle). and gap interacts 2005), that red directly regulates ZO-1 report 8A, so, that (Fig. to known doing function first in and the and positioning ZO-1, is with study interacts interacts This directly Vcl ZO-1. that and with myocytes thin- cardiac in a forms complex with presenting effusions. also pericardial E10, and myocardium by global walled that die mention Vcl embryos to our important with Vcl-knockout is compare postnatal It to in data. useful knockout ZO-1 be cardiomyocyte would of ZO-1 role of cardiac-myocyte-specific- studies the use knockout that address this studies to Future Therefore, used myocytes. 2008). cardiac be al., not et could (Katsuno model E11.5 by lethality in gestation (suggesting apparent 2009). effusions al., not pericardial et interaction (Li manner is Cx43 cell-type-specific a interaction the in occurs that this ZO-1 with suggesting 1998; 1998), epithelium, Moolenaar, al., and seminiferous (Giepmans et myocytes cardiac Toyofuku binds in directly Cx43 disc Cx43- ZO-1 Whereas intercalated 8A). to 2012). regulate (Fig. Liang, to and discs (Delmar known function intercalated are junctions at Gap gap found 8A). containing also (Fig. as sites are adhesion junctions junctions cell-to-cell cell-to-matrix cadherin-based integrin-containing as in that well states present literature is Current Vcl cells. adjoining between connection a ucinfnto,wudb etre Fg 8B). ultimately, cardiac (Fig. and perturbed the be junction, in would gap function, present the junction were in gap positioning or Vcl Vcl Cx43 wild-type mutant myocyte, a of if that amount suggests reduced here data together, Taken circle). odcags eaiet AD,wr oe npopoyae PI3K phosphorylated in noted were GAPDH, to relative changes, fold (4.00 P P u oe aapeetdhr niaeta Vcl–ZO-1–Cx43 a that indicate here presented data novel Our angiogenesis, abnormal in results mice in ZO-1 of deletion Global , , 6 .5.(,)Cmae ocnrlcls h odcag fteautophagy the of change fold the cells, control to Compared (E,F) 0.05). 6 6 6 .0) CD h c soitdproteins associated Vcl The (C,D) 0.001). .9 C yeI 0.61 I, Type LC3 0.09, .5,a ela h ppoi akr AP(0.25 PARP markers apoptotic the as well as 0.05), .3 * 0.03, .23% ** 32%, 0.32 b P -nei rti xrsin ugsigta c sa is Vcl that suggesting expression, protein 1-integin , .5 n TN(0.60 PTEN and 0.05) P 6 , 0.07,** .0)adAkt and 0.001) 6 2 ti nw htP3 euae the regulates PI3K that known is It . .1,rltv ocnrl l aus* values All control. to relative 0.11), 6 P .3 n C yeI,0.47 II, Type LC3 and 0.13, , .0)adpR2tR2(4.65 pERK2/tERK2 and 0.001) b -nernadC4 Fg A red 8A, (Fig. Cx43 and 1-integrin S473 6 nutero in .1 * 0.11, P tk (5.94 /tAkt 6 , .7fl,rltv ocontrol to relative fold, 0.17 .0) H rgtfedimages field Bright (H) 0.001). P a a , -actinin, er alr)admid- and failure) heart ctnn(Mu -catenin fl .5 rtis naddition, In proteins. 0.05) / fl 6 MM,rdcinin reduction NMVMs, .8 *** 0.08, AB etr blot Western (A,B) 6 b .6.Decreased 0.06). 6 ctnnand -catenin .4 and 0.04) P P le tal., et ¨ller 6 , , 0.06, 0.0001), 0.001, T308 (Y458) /tAkt W ta. 02.Arcn td ecie htitreec with interference cells that all describes in study signaling recent maintained A Akt1 2012). brain, activity al., caspase-3 mouse inhibited et and in (Wu (Ching activity, inhibition other PTEN starved decreased kinase in but serum Rho Akt were shown 2010). cells al., been muscle et also skeletal a has where as studies phosphorylation activation deficiency Akt Akt function. 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Journal of Cell Science oso elt-arxitrcin Mce,20) Weakened 2003). (Michel, interactions cell-to-matrix of loss cardiac of survival for Anoı necessary of myocytes. is matrix 2012). rate extracellular membrane glycoprotein to al., Rho structural binding et through enhanced the adhesions (Aguilera initial cellular function of of an actin Maintenance this members the regulate require different tightly family cells and in formation, when autophagosome participates stage remodeling cytoskeleton actin ARTICLE RESEARCH ihdltda ela yetohchmncardiomyopathy human hypertrophic as associated be well to as known are MetaVcl dilated Vcl isoform and splice-variant familial with 2009). MVcl its Ca in and al., in Vcl Mutations et is cytoskeletal, (MVcl). involved genes al., (Hershberger these structural, of proteins et be One mitochondrial (Morita to affect and failure cycling known also heart linked currently developing cardiomyopathies been Genes of have humans risk 2005). in increased proteins Z-disc- to desmosomal metabolic, sarcomeric, and of related Na mutations Delmar, and and Single-gene adhesions (Agullo-Pascual 2012). cell proposed been 2011). intercellular has in al., function and Cx43 et channel for O’Quinn control gap rapid role 2011; to a al., targets is Recently, therefore et pharmacological (Colussi communication and new arrhythmias as cardiac junctional propagation, studied are gap potential junctions of action role coordinated arrhythmias. towards key heart myocyte the These A predispose cardiac and diminished sites. function, to to heart attachment lead and owing might junctional thereby integrity sites gap contact adhesions, membrane impaired and of cell integrin reduction connexin-based weakened the as to well reduction a contributing as triggers Vcl integrin diminished that in shows study current The Implications purpose. this one. for mutant required the is with expression work protein the myocyte Future replace wild-type ZO-1 to respective ZO-1. and used the and be of Vcl Vcl could of mutants mutant function other these of any Ultimately perturb production not Vcl–ZO- do and that the constructs of domain(s) localization necrosis. precise binding myocyte more mechanism cardiac 1 require would only or so transduction all the do signal communication, To is in junction gap changes binding of the ZO-1–Vcl perturbation of for responsible loss that prove techniques. isolation cell standard fragile to extremely intolerant are cells and mice) these Vcl-deficient cVclKO because use our analysis, from to biochemical derived for able are ones for not which (e.g. myocytes were myocytes, cardiac necessary We cardiac adult of cells. neonatal some adult of is from that use clearly different on was expression data relied limitation study our further Vcl our A that physiological integrity. believe cardiomyocyte We that cells. to shows a healthy Vcl investigate to of of important zero. population was reduction It essentially resulted. with changes was morphological even that model expression myocyte , found protein a we in Vcl Vcl Unfortunately, studied where have would system study our Ideally, limitations Study inhibits reduction with cells Vcl to expression. compared Vcl that anoı signaling physiological apoptotic, suggest as during also well as here occur might autophagic, data can membrane and Our to susceptible which sarcolemma rupture. extremely are death, the myocytes cardiac cell to Beating necrotic damage induce induce sites adhesion 0 fnra hsooia ees rfudbohmcland biochemical profound levels, physiological normal of 50% iiaino u auciti htw antabsolutely cannot we that is manuscript our of limitation A ksi eie sporme eldahoigto owing death cell programmed as defined is ¨kis ¨kis. 2+ + 98.HwZ- sicroae notebreso h gap the of al., borders et the Toyofuku into 2011; incorporated al., 2005; is et al., ZO-1 Rhett et How 2012; Hunter 1998). Gourdie, 1998; prior and several Moolenaar, in Rhett and stabilization and (Giepmans that function studies junction shown gap in player have cardiomyopathy mice 2007). dilated al., or et cVclKO arrhythmias expression, (Zemljic-Harpf lethal Cx43 either in induced altered and reduction studies Vcl cardiomyocyte-specific al., Our et that (Zemljic-Harpf stress and 2004). hemodynamic structure to disc shown intolerant intercalated has were altered they cardiomyopathy-linked mice had a VclKO animals as hemizygous these These that Vcl using 2013). work of Previous al., role gene. the induce et illustrate could (Tangney all sarcomeres thereby reports in and stiffness, stress systolic spacing, mechanical increased interfilament deficiency increasing Vcl by that shown been observed has been has as discs well intercalated as after at costameres, Vcl a of at because alignment expression irregular relevant Vcl is of This reduction significant arrhythmias. (Maeda and cardiomyopathy dysfunction 2006b; dilated cardiac addition, al., in with In absent et 1997). patient be al., Vasile to a et found 2006a; of was tissue al., Vcl Furthermore, cardiac et 2006c). 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Basso molecular lethal by the develop cardiac to proposed prone as heart in are Therefore, end-stage hearts in communication these reduced and junctional is failure, expression gap junction Gap proper myocytes. ensure and in in junctions important tight and be adherens tissues. gap, could many of here function the discussed modulating interaction Vcl–ZO-1 tsol entdta O1hsbe hw ob key a be to shown been has ZO-1 that noted be should It nsmay edmntaehr o h is ieta ZO-1 that time first the for here demonstrate we summary, In enhance to target new important an as Vcl describe data Our a rpnsm cruzi Trypanosoma ctnn ihti,w ol ugs httenovel the that suggest would we this, With -catenin. ora fCl cec 21)17 1411 doi:10.1242/jcs.143743 1104–1116 127, (2014) Science Cell of Journal neto Ml ta. 04.Rcnl,it Recently, 2004). al., et (Melo infection rpnsm cruzi Trypanosoma neto a edto lead can infection a -actinin, 1113 b 1D

Journal of Cell Science icswr ietdoengti 0m f05m/ltysnHS at trypsin-HBSS ventricular mg/ml the 0.5 then of ml and 50 removed in 4 were overnight digested atria were the pieces excised, were hearts hsspraat eas nynnmoyeclsrpdyahr othese 5 to Approximately adhere rapidly dishes. cells from on non-myocyte uncoated NMVMs only h each purify because 1 further supernatant, for to this used each terminated were steps, cells dishes, pre-plating and tissue-culture Two collagenase-II uncoated pooled centrifugation. were by inactivate digests serum- collected were from ice-cold Supernatants to Adding digestion. min. collagenase 5–7 medium for each (200– containing collagenase digestions, for II NJ) additional type Lakewood, three Corporation, in solution Biochemical continued Worthington medium was Units/ml, 300 free agitation digestion gentle and trypsin/serum with removed the was minutes Subsequently 3–4 rpm). for digested (100–150 and medium serum-free muoirsoi nlsso ye o rprto fprotein. for of acetone with preparation fixed for either and lysed 2007). h al., or 4 et for O’Connell analyses 2012; cultured wild-type al., immunomicroscopic were et 2–3-month-old cells (Li from described These facility. isolated previously AALAC-approved as were an mice in myocytes housed cardiac use the were and Adult mice with care animal all accordance institutional and the in committee, by approved conducted guidelines, were care animal procedures experimental culture The and isolation myocyte Cardiac METHODS AND MATERIALS ARTICLE RESEARCH 1114 (5–7- as well sections as tissue NMVMs, cardiac and adult frozen cultured studies, immunofluorescence For analyses immunoblotting and immunoprecipitation Immunofluorescence, Vcl the plating after days cultures Five NMVM of infection Adenoviral 0–2- form isolated were (NMVMs) Vcl myocytes day-old cardiac mouse Neonatal otiigmdu o 4h n hnmdarpae ihserum- with in replaced outlined daily. as media replaced periods media then for new continued with and results, were h, Cultures 24 medium. containing for or medium Cre-recombinase containing either MOI expressing at LacZ, (AdV) adenovirus recombinant ah6-mclueds,wihwr otdwt .%gelatin/ 0.2% with coated were which dish, (1 culture fibronectin 60-mm each iulzduigceiuiecn usrt o h eeto of ( detection and digitally the performed below, was for listed quantitation (Pierce substrate Densitometric as peroxidase chemiluminescent antibodies, horseradish using primary visualized available commercially ctn o i n ahdwt B.Atrbokn o 0mnat 4 min at 30 (overnight for stained blocking were After tissues PBS. or with cells washed temperature, and room min 7 for acetone MET,1 excoi cd .%SS %N-0 n equal and NP-40, 1% SDS, 0.2% NaCl, mM acid, (5 150 deoxycholic amounts 7.5, protein pH 1% Tris-HCl EDTA, mM mM 50 1 containing buffer previously in lysed as were CA)], 2004). 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Journal of Cell Science ri,S .adPro .V. J. Pardo, and W. S. Craig, ot,C n ish E. Hirsch, and C. Costa, S., Rossi, D., Stilli, R., Berni, F., Spallotta, S., Straino, J., Rosati, C., Colussi, S. J. Fisher, and S. Banerjee, N., Marupudi, P., Rajguru, K., J. Ching, hta,M .&Hno,C V. C. Hinton, & A. M. M. Chetram, B. Jockusch, F., Entschladen, R., M. Holt, E., T. Stradal, T. S. I., Izard, L. Chandrasekar, Musil, and and R. C. P. A. Le, Bois, A., G., B. Boswell, Bricogne, C., Vonrhein, A., R. Borgon, nlzda rvosydsrbd(owl ta. 09 lFuye al., 2000). Rivedal, et and and el-Fouly loading, Opsahl 2009; 2011; dye al., al., transfer after et et Dye min (Boswell Li loading. 25 1987; described dye acquisition previously to image additional as prior by an analyzed saved for assessed medium incubated then They culture and scratch. was the PBS, the in with flooding min times by 20 min three 2 rinsed for then and dyes Cells were Yellow dextran Lucifer monolayer. Rhodamine mM mM confluent 1 containing 1 the PBS with though loaded immediately scratch were longitudinal a generate ARTICLE RESEARCH os .R,OHr,B . itipc,D,Krptik .adIad T. Izard, and J. Kirkpatrick, D., Nietlispach, P., B. O’Hara, G. R., Thiene, P. and Bois, D. Corrado, B., Bauce, C., A. Basso, J. DeCaprio, and H. S. Ali, Bero O., M. Aguilera, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.143743/-/DC1 online available material Supplementary material Supplementary months. in 12 Deposited American after A.E.Z.-H.]. the release to and 2260359 for R.S.R.]; AHA-BGIA PMC to number PO1 HL103566 [grant numbers RO1 Association [grant and Heart Health HL088390 of RO1 Institutes National 46345, the HL by supported was work This Funding the composition. writing figure design, final overall experimental to guiding and the and for to studies, manuscript responsible contributed two-hybrid was yeast direction, A.E.Z.-H R.S.R. out research with revision. carried together and manuscript 8 designed provided figure A.A.D. in A.R.B. R.S.R. representation cryosectioning. and performed diagrammatic and the maintained colonies, generated Lucifer E.K.A. mouse and revision. and genotyped Dextran manuscript studies, provided and Rhodamine and immunoblotting acquired data, P. and microinjection J.C.G. O. culture Yellow figures. revision. tissue the and of of manuscript manuscript most execution provided the of the of design to and revision contributed and execution drafting the for experiments, responsible was A.E.Z.-H contributions Author interests. competing no declare authors The scientific interests their Competing for CA) Jolla, and La IL.) Medicine, of Chicago, protocol. School support. Illinois-Chicago, isolation (UCSD (U. myocyte discussions Yuan Marsala helpful Jason neonatal Martin for the acknowledge and CA) optimizing further NY) Jolla, in We York, La advice New Medicine, technical Medicine, of and of School School (UCSD Sheikh (NYU Farah Mezzano Valeria thank We Acknowledgements gloPsul .adDla,M. Delmar, and E. Agullo-Pascual, iaetpoen ooaiewt iclna otmrs myofibril-to- costameres, at vinculin with sites. attachment colocalize sarcolemma proteins filament fPI3K. of and normal in 43 connexin of heart. lateralization al. and dystrophic et junctions A. GAP from Mai, dissociation E., Macchi, E., Musso, starvation. serum after action insulin 299 increased in AMPK for role cancer. turnover. H. adhesion-site W. Ziegler, and cells. lens in communication junctional gap of vinculin. human of structure Crystal h iclnbnigstso ai n lh-cii r ufcett activate to sufficient are alpha-actinin and talin of sites vinculin. binding vinculin The cardiomyopathy. arrhythmogenic proteins. host with interaction antigen: nteheart. starvation the upon in formation autophagosome of events autophagy. early induced the in participates C1171-C1179. , eetSga rndc Res Transduct Signal Recept J ur o.Mcoil Immunol. Microbiol. Top. Curr. .Bo.Chem. Biol. J. .Mmr Biol. Membr. J. rc al cd c.USA Sci. Acad. Natl. Proc. ,W n oob,M I. M. Colombo, and W. n, ´ Autophagy 20) iclnat sasno nlpdrglto of regulation lipid in sensor a as acts Vinculin (2005). .Cl Sci. Cell J. 281 21) oeta utknss h cfodn function scaffolding the kinases: just than More (2010). 7228-7236. , 245 18) am ci,seti,adintermediate and spectrin, actin, Gamma (1983). 20) ellrtasomto yS4 ag T large SV40 by transformation Cellular (2001). elMotil. Cell 21) TNrglto fEK/ inln in signaling ERK1/2 of regulation PTEN (2012). 8 477-482. , 1590-1603. , a.Rv Cardiol. Rev. Nat. 21) h ocnnclfntoso Cx43 of functions noncanonical The (2012). 118 Structure 21) N (2011). 1461-1472. , ei.Cne Biol. Cancer Semin. 32(4) 346 20) peuainadmaintenance and Upregulation (2009). 3 449-462. , 171-181. , 108 190-195. , 12 x.EeRes. Eye Exp. e 21) h ci cytoskeleton actin The (2012). 1189-1197. , lsn ctlto determines acetylation -lysine 2795-2800. , 21) ahpyilg of Pathophysiology (2011). 9 223-233. , 11 88 15-23. , m .Physiol. J. Am. 919-927. , 21) A (2010). (2004). (2006). iPoo .adD ail,P. Camilli, De and G. Paolo, Di X. F. Liang, and M. Delmar, erc,E,Vne,M,D us,E,Kyk,D . ’ed,K,Vanhaecke, K., D’Herde, V., D. Krysko, E., Vuyst, De M., Vinken, E., Decrock, utr .W,Bre,R . h,C n ori,R G. R. Gourdie, and C. Zhu, J., R. Barker, W., A. Hunter, M. C. Waterman-Storer, and G. Danuser, T., K. Applegate, L., Ji, K., Hu, esbre,R . idned . etoi . eda,C . alr .R., M. Taylor, E., C. Seidman, L., Mestroni, J., Lindenfeld, E., R. Hershberger, M. J. Anderson, and S. A. Fanning, utr .W n ori,R G. R. Gourdie, and W. A. Hunter, rsof . ofa,B . rne,M . hu . asn,M,Yang, M., Parsons, R., Zhou, D., M. Brenner, D., B. Hoffman, C., Grashoff, K. Burridge, and P. A. H. Gilmore, W. Moolenaar, and N. B. Giepmans, M. J. Anderson, and Y. T. Ma, S., A. Fanning, C. C. Chang, and E. J. Trosko, H., M. El-Fouly, Zamir, A., C. Gersbach, J., Scrimgeour, A., Singh, T., T. Lee, W., D. Dumbauld, nk,A,Io,M,Tmr,A,Mtua . uue .adTuia S. Tsukita, and M. Furuse, M., Matsuda, A., Tamura, M., Itoh, A., Inoko, i . a,J,Si . aaa . h,Y,Yn . ase . Kitamura, H., Matsue, Q., Yan, Y., Chi, N., Sawada, L., Shi, J., Yao, K., Li, J. Schaper, and D. Scholz, E., Arnon, S., Hein, S., Kostin, Waterfield, and J. Timms, M., S., White, Jeyaraman, K., Ahmadi, E., K., B. Okkenhaug, R., Nickel, Katso, A., D. Iacobas, X., Dang, E., Kardami, A. D. R. Goodenough, P. and Bois, A. and L. G. Jesaitis, Bricogne, L., C. Rush, A., R. Borgon, G., Evans, T., Izard, hg,A,Mrlo . eio .adHrc,E. Hirsch, and A. Perino, F., Morello, A., Ghigo, siaa . uo . an,M,Mk,T,Kuu . th .adMua T. Miura, and T. Itoh, H., Kouzu, T., Miki, M., Tanno, A., Kuno, S., Ishikawa, i .W,Mu,D . e,W .adCeg .Y. C. Cheng, and M. W. Lee, D., D. Mruk, W., M. Li, and E. M. Weber, L., N. Malinin, L., Ding, Z., X. West, L., Ma, A., B. Kerr, K., Takeuchi, M., Itoh, A., Tamura, M., Hata, T., Matsui, K., Umeda, T., Katsuno, wtt,M,G,Y,Deel,T,Iaaa . eesn .L,Hsiia M., Hoshijima, L., K. Peterson, Y., Iwanaga, T., Dieterle, Y., Gu, M., Iwatate, icfunction. disc inln ncl et:t ieo e die? let or live to L. death: Leybaert, cell and in V. signaling Rogiers, P., Vandenabeele, T., adoypty–aHatFiueSceyo mrc rcieguideline. practice America of Society Fail. Failure Heart a – cardiomyopathy A J. Towbin, cldn- lescnei4 a ucinsz n raiainb influencing by organization and accretion. size channel junctions. junction gap gap connexin43 alters 43 occludens-1 connexin to targeting for Adhes. Commun. dispensable is occludens-1 adhesions. focal within motion actin 111-115. of transmission Differential ehncltninars iclnrvasrglto ffcladhesion focal of regulation reveals al. et vinculin T. Ha, S., across dynamics. C. Chen, tension G., S. Sligar, mechanical A., M. McLean, T., M. occludens-1 zona phosphatidyl-inositol-4-5-bisphosphate. the by actin of domain PDZ second the protein. with interacts connexin43 junctions. cellular of intercellular assembly the junctional Sci. Acad. regulate gap that study scaffolds cytosolic to technique simple and communication. rapid A al. et transfer. W. S. Craig, E., J. Curtis, transmission. S., force C. regulates Chen, J., Fu, A., E. yoieidcdidcbeN ytae(NS n onxn3i bladder in connexin43 and (iNOS) cells. synthase muscle smooth NO inducible M. cytokine-induced Takeda, and M. cancer. and homeostasis, development, for D. M. expression. C. gene and D. growth Spray, Biol. cell and controlling in S. Tanguy, connexins of J., Makazan, the W., and Srisakuldee, ZO-1 to homologous protein. protein suppressor junction tumor tight discs-large a Drosophila ZO-2, of distribution conversion. tissue bundle helical through talin by 427 activation Vinculin (2004). heart. mouse adult the in 1814-1820. switching gene Cre-LoxP and delivery iae nhat n disease. ZO-1. and protein health junction in kinases tight the in region binding J. actin FASEB the of characterization dynamics. membrane 21) oeo onxn4 npoetv IKAtGK3easgaigin signaling PI3K-Akt-GSK-3beta protective in connexin-43 of cardiomyocytes. Role mouse (2012). in protein, MAGUK junction tight a ZO-3, tissues. of distribution and Expression lkpii- sapoencmlxta euae lo-etsbrirdynamics. barrier USA blood-testis Sci. regulates Acad. that Natl. complex Proc. protein a as plakophilin-2 heart. failing human the in 271-280. proteins related and cytoskeleton stress. oxidative of consequences the 6(287) limiting by death and infarction V. T. Byzova, cells. embryonic of yolk al. apoptosis defected 2475. with and et associated angiogenesis T. phenotype sac lethal Noda, embryonic Y., causes occludens-1 Nabeshima, T., Fujimori, he,K .adRs,J. Ross, and R. K. Chien, 171-175. , 15 94 a7 o:6/287/ra67 doi: ra67. , 83-97. , 20) ellrfnto fpoponstd -iae:implications 3-kinases: phosphoinositide of function Cellular (2001). 245-264. , ora fCl cec 21)17 1411 doi:10.1242/jcs.143743 1104–1116 127, (2014) Science Cell of Journal ur Biol. Curr. ee Cells Genes 16 Nature 1165 1835-1837. , ;HatFiueSceyo mrc 20) eei vlainof evaluation Genetic (2009). America of Society Failure Heart .; 21) nefrnewt k inln rtcsaantmyocardial against protects signaling akt with Interference (2013). er Rhythm Heart 113-120. , x.Cl Res. Cell Exp. mJPyilHatCr Physiol Circ Heart Physiol J Am 15 466 o.Bo.Cell Biol. Mol. 8 931-934. , 55-63. , 8 .Bo.Chem. Biol. J. 21) eirclrglto ewe proinflammatory between regulation Reciprocal (2011). 263-266. , Nature 837-845. , 21) onxn3adterglto fintercalated of regulation the and Connexin43 (2012). 106 20) nvv ihefcec rncrnr gene transcoronary high-efficiency vivo In (2003). 9 rc al cd c.USA Sci. Acad. Natl. Proc. 443 835-838. , 19) euaino iclnbnigt ai and talin to binding vinculin of Regulation (1996). 20) hshioiie ncl euainand regulation cell in Phosphoinositides (2006). ucl.Biochem. Subcell. 10213-10218. , 168 651-657. , 16 20) h eodPZdmi fzonula of domain PDZ second The (2008). 422-430. , 5686-5698. , 286 20) ouaocues1ad- are -2 and occludens-1 Zonula (2009). 19) oeua hrceiainand characterization Molecular (1994). 41552-41562. , elDahDiffer. Death Cell nuRvCl e Biol Dev Cell Rev Annu 19) h a ucinprotein junction gap The (1998). 18) caelaigaddye and Scrape-loading (1987). 20) slto n functional and Isolation (2002). Nature 20) eiinyo zonula of Deficiency (2008). .Cl Biol. Cell J. 302(12) 58 21) hshioiie3- Phosphoinositide (2012). 183-213. , 20) onxn4 and 43 Connexin (2009). 20) Connexin-related (2009). o.Bo.Cell Biol. Mol. 381 110 H2536-2544. , 21) o vinculin How (2013). 16 rg ipy.Mol. Biophys. Prog. 531-535. , er al Rev. Fail. Heart 21) Measuring (2010). 9788-9793. , 524-536. , 124 20) h role The (2007). 20) Zonula (2005). eeTher. Gene Science 949-961. , 17 20) The (2000). n.N Y. N. Ann. c Signal Sci 615-675. , 19 .Card. J. 2465- , (2007). (2003). Nature 1115 315 Cell 10 5 , , ,

Journal of Cell Science anes .M,Hl,M . enns . utn .H,Bruo,I L., I. Barsukov, H., D. Sutton, L., Jennings, R., M. Holt, M., A. R. K. Yamada, Saunders, and G. J. Laing, E., J. Saffitz, G. R. Gourdie, and J. Jourdan, M., J. Rhett, igi .adGmn,M. Gimona, and V. Niggli, ht,J .adGude .G. R. Gourdie, and and M. C. J. J. Rhett, Loftus, Y., S. Shai, T., H. Vu, S., R. Keller, E., A. Harpf, G., C. Pham, Peng,X.,Cuff,L.E.,Lawton,C.D.andDeMali,K.A. W. S. Craig, and D. J. Siliciano, V., J. Pardo, i . u . as,A . u . io . sal,S,Yjm,T,Nue,U., Nguyen, T., Yajima, S., Israeli, P., Liao, Y., Gu, M., A. Manso, Y., Wu, R., Li, ARTICLE RESEARCH 1116 G. R. Gourdie, and W. K. Hewett, S., B. Harris, A., J. Palatinus, P., M. O’Quinn, C. P. Simpson, and C. M. Rodrigo, D., T. O’Connell, M. M. Burger, and J. Brunner, P., D. Dimitrov, V., Niggli, E. Mu C. Seidman, and J. Seidman, H., Morita, B. J. Michel, amr .M,Pafr,M . ri,S . calr .D n apel .L. S. Campbell, and D. M. Schaller, W., S. Craig, P., M. Playford, M., S. Palmer, E. Rivedal, and T. H. M. Opsahl, Keating, S., Huttelmaier, Y., N. Kishimoto, S., Illenberger, M., T. Olson, eo .G,Amia .S,d erle,M .adPria .C. M. Pereira, and N. M. Meirelles, de S., D. Almeida, G., T. Melo, Woodco F., Amirahmadi, R., J. McMullen, H. W. A. Dillmann, and K. X. J. DeMali, Yuan, J., and Suarez, O., S. Platoshyn, A., A. Makino, Fanning, X., Peng, L., D. R. J. Bies, Maiers, and S. Valent, B., Lowes, E., Holder, M., Kiriazis, Maeda, C., B. Bernardo, B., R. Williams, M., X. Gao, L., K. Weeks, C., R. Lin, lr .L,Prwc,M,Shit . tpegnv .I,Hbr . lsg .E. I. Blasig, O., Huber, I., D. Utepbergenov, A., Schmidt, M., Portwich, L., S. ller, ¨ okv .adCicly .R. D. turnover. Critchley, adhesion and A. Bobkov, excitability. cardiac for implications : turnover occludens-1. zonula by regulated is 1516-1528. transition junction gap ucinorganization. junction pathway. response hypertrophic myocyte 279 cardiac in involved S. R. Ross, beta-catenin. to binding by expression E-cadherin surface (‘‘costameres’’) elements sarcolemma. lattice and USA myofibrils transverse Sci. between muscle: attachment of skeletal sites mark in lattice cortical 21) etd iei ftecnei4 abxltriu eue gap injury. reduces ventricular terminus following carboxyl arrhythmia connexin43 induced the and of remodeling mimetic junction peptide A (2011). a myocytes. cardiac with mouse adult analyzed of structures bilayer with vinculin phospholipid. photoactivatable component cytoskeletal the bilayers. 213 acidic-phospholipid and (meta)vinculin alpha-actinin, ZO-1. with mechanism interaction common a Chem. share alpha-catenin protein G. Krause, and failure. heart mediators. extracellular al. et D. N. Dalton, S., M. Huang, 20) ii idn oteti oano icln pcfct n h oeo the of role the and specificity vinculin: termini. of C domain tail and the N to binding Lipid (2009). analysis. image and loading Commun. scrape by communication intercellular cardiomyopathy. M. dilated B. Jockusch, and Res. rpnsm rz neto irpsvnui otmrsi cardiomyocytes. in costameres Biol. vinculin Cell J. disrupts Eur. infection cruzi Trypanosoma L. G. Jennings, cardiomyopathy. 3-kinase(p110alpha) and A. phosphoinositide K. and Hewitt, D., cell R. endothelial coronary with associated mice. C230. diabetic is streptozotocin-induced in connexin40 dysfunction of Downregulation junctions. adherens to junctions to recruitment defi with metavinculin. associated and miRNA cardiomyopathy PI3K-regulated of identification failure: mRNA. heart R. infarction-induced J. McMullen, and H. responses. signaling Pathol. and J. mechanical defective Am. causes myocyte cardiac murine H2916-H2926. , 1009-1015. , 108 280 reislrTrm acBiol Vasc Thromb Arterioscler 704-715. , 80 7 3747-3756. , 20) nii ntecrivsua ytm nw n unknown and known system: cardiovascular the in Anoikis (2003). 367-375. , 1008-1012. , .Ci.Invest. Clin. J. Circulation 20) titdmsl-pcfcbt(D-nernadFKare FAK and beta(1D)-integrin muscle-specific Striated (2000). a 180 20) h ih ucinpoenocui n h deesjunction adherens the and occludin protein junction tight The (2005). ctnnanvlmcaimfrculn h sebyo tight of assembly the coupling for mechanism novel -catenin—a rcNt cdSiUSA, S U Sci Acad Natl Proc 83 .Bo.Chem. Biol. J. 952-962. , u elBiol Cell J Eur 531-540. , er Rhythm Heart 20) eaiclnmttosatratnitrcinin interaction actin alter mutations Metavinculin (2002). reislr hob ac Biol. Vasc. Thromb. Arterioscler. Circulation 21) IKp1 lh)poet gis myocardial against protects alpha) PI3K(p110 (2010). 95 19) vdnefratrayitrcinbetween interaction ternary a for Evidence (1993). 17-20. , 20) uniaiedtriaino a junction gap of determination Quantitative (2000). 115 21) h eieu:anwfaueo x3gap Cx43 of feature new a perinexus: The (2012). .Bo.Chem. Biol. J. 518-526. , 284 .Cl Sci. Cell J. 5(6) 85 (2012). ehd o.Biol. Mol. Methods 20) oeo iclni euaigfocal regulating in vinculin of Role (2006). 105 7223-7231. , 9 619-623. , k .A,ShneBan . Bouwman, M., Schinke-Braun, A., E. ck, 30(4) 8-0.di S0171-9335(06)00025-2 doi: 487-500. , 431-437. , inyo h yokltlprotein cytoskeletal the of ciency 104(2) b inln ndltdadhypertrophic and dilated in signaling 20) rtcieefcso exercise of effects Protective (2007). nerngn xiini h adult the in excision gene integrin 1 2-3.di 30/4/724 doi: 724-732. , 126 20) eei asso human of causes Genetic (2005). 21) onxn4 onxnto connexon 43 Connexin (2011). 20) onxnepeso and expression Connexin (2000). ic Res. Circ. 261 3904-3915. , 612-617. , 18) vinculin-containing A (1983). 6912-6918. , 21) iclnrgltscell- regulates Vinculin (2010). 20) slto n culture and Isolation (2007). m .Physiol. J. Am. 357 .Cl Sci. Cell J. 86 23 18) neato of Interaction (1986). 271-296. , 723-728. , 2146-2154. , o.Bo.Cell Biol. Mol. rc al Acad. Natl. Proc. u.J Biochem. J. Eur. m .Physiol. J. Am. 19) Dilated (1997). 21) ZO-1 (2013). 123 elAdhes. Cell 295 ,567-577. C221- , (2004). (2008). .Biol. J. Circ. 22 , uase .C,Prz . dmo,E . unr .E,Jne,S n Hahn, and S. Junger, E., C. Turner, D., E. Adamson, O., Pertz, C., M. Subauste, aie .C,Ewrs .D,Omn .R n cemn .J. M. Ackerman, and R. S. Ommen, D., W. Edwards, C., V. Vasile, R. P. Brink, and C. M. E. Beyer, Tada, V., and Valiunas, M. Hori, T., Kuzuya, K., Otsu, M., Yabuki, T., Toyofuku, ige,W . idntn .C n rthe,D R. D. Critchley, and C. R. Liddington, H., W. Ziegler, og .S,Gaioi,S,GmzVqe,L,Sbe .A,Zmn A., Ziman, A., E. Sobie, L., Gomez-Viquez, S., Guatimosim, S., T. T. L. Hong, Song, S., T. Fong, S., S. Zhang, J., P. Buch, M., J. Vogan, Terracio, W., J. T., Smyth, Hirozane, R., Price, W., Carver, G., D. and N. Simpson, Y. Jan, I., M., Shiraishi, Zastrow, von A., M. Puthenveedu, J., A. Fay, M., R. Shaw, izu . irao .J n ios M. Simons, and J. F. Giordano, P., D., Tirziu, Liao, A., Krishnamurthy, S., M. Janssen, S., J. Chuang, R., J. Tangney, M. K. Hahn, and D. E. Adamson, P., Nalbant, C., M. Subauste, ek,K . a,X,D,X . oy .J n asmt,A Bernardo, A, Matsumoto, and J. E. Boey, J., X. Du, X., Gao, L., K. Weeks, R. D. Critchley, and T. S. Barry, J., Weekes, J. M. Ackerman, and D. W. Edwards, R., S. Ommen, C., V. Vasile, eli-ap,A . ilr .C,Hnesn .A,Wih,A . as,A M., A. Manso, T., A. Wright, A., S. Henderson, C., J. Miller, E., A. Zemljic-Harpf, P., K. Roos, C., M. Jordan, T., R. Avalos, S., Ponrartana, E., A. Zemljic-Harpf, i,H .adJne,P A. P. Janmey, and L. H. Yin, D. E. Adamson, and H. Baribault, D. W., Xu, Wu, and Y. Fang, P., Liu, H., Hu, J., Li, J., Wu, Ru M., Kroemker, E., E. Weiss, and M. T. Olson, D., W. Edwards, R., S. Ommen, L., M. Will, C., V. Vasile, .M. K. hwqatttv ifrne nselectivity. in differences quantitative cardiac show in ZO-1 with connexin-43 protein junction gap myocytes. the of association Direct euaino vinculin. of regulation 7522-7537. atan .adLdrr .J. heart. W. in Lederer, tubules and H. Hartmann, membrane. plasma the to vesicles 43 989. connexin of M. traffic R. Shaw, and myocytes. cardiac mouse 29 fetal K. in T. arrangement myofibrillar Borg, and junctions. L. adherens to interior cell the from directly Y. L. Jan, btutv yetohccrimoah sascae ihrdcdexpression reduced with associated is cardiomyopathy hypertrophic Obstructive heart. Zemljic-Harpf, M., Muthuchamy, A., dysfunction. G. al. Meininger, et X., A. Wu, adherens M., the Hoshijima, of interaction the MAGI-2. protein maintaining scaffolding 280 the by with beta-catenin level protein junction protein PTEN controls motility. and survival control samse euao feecs-nue adortcinadP3 gene PI3K and cardioprotection dysfunction. exercise-induced cardiac of R. rescues regulator J. master therapy McMullen, a of and is sites. regions C. phosphorylation B. C-terminal C and kinase N- protein and J. the Biochem. actin-binding between exposing association vinculin, intramolecular the disc. intercalated 715. the in vinculin of ucin,cuigsde et rdltdcardiomyopathy. al. dilated cellular et or D. disrupts A. death gene McCulloch, sudden S. vinculin A., causing the G. junctions, R. of Perkins, K., excision Ross, A. Cardiac-myocyte-specific Thor, (2007). D., and N. Dalton, D. L., stress-induced Elsherif, E. to predisposes Adamson, gene Q., vinculin V. the cardiomyopathy. of Phan, inactivation D., Heterozygous N. Dalton, cytoskeleton. aui,Peet ernlAotssvateAtAtvto n PTEN and Activation development. embryonic Akt during defects brain the Brain. and Rat via of Penumbra Apoptosis Ischemic the in Neuronal Inactivation Prevents Fasudil, vinculin. and alpha-catenin between formation M. CIRCHEARTFAILURE.112.966622 cardiomyopathy. dilated and hypertrophic Metab. both Genet. with associated R975W, confers J. M. vinculin, Ackerman, protein, expressed cardiomyopathy. ubiquitously hypertrophic Commun. a to in susceptibility mutation missense 2041-2052. , 19) iclni ato h ahrnctnnjntoa ope:complex complex: junctional cadherin-catenin the of part is Vinculin (1998). 5676-5681. , 20) iclnmdlto fpxli-A neatosrgltsEKto ERK regulates interactions paxillin-FAK of modulation Vinculin (2004). Circulation ora fCl cec 21)17 1411 doi:10.1242/jcs.143743 1104–1116 127, (2014) Science Cell of Journal 21) oe oefrvnui nvnrclrmoyemcaisand mechanics myocyte ventricular in vinculin for role Novel (2013). 345 .Bo.Chem. Biol. J. 20) irtbl lsedtakn rtistre a junctions gap target proteins plus-end-tracking Microtubule (2007). 314 ipy.J. Biophys. nu e.Physiol. Rev. Annu. 998-1003. , 87 827-832. , n cdSci Acad Y N Ann m .Pathol. J. Am. 169-174. , 122 20c.Ietfcto famtvnui isnemutation, missense metavinculin a of Identification (2006c). 21) ci yokltnrs tp euaeanterograde regulate stops rest cytoskeleton Actin (2012). 928-937. , 19) iclni nesnilcmoetfrnormal for component essential an is Vinculin (1997). rnsCl Biol. Cell Trends 104 273 1623-1633. , .Cl Biol. Cell J. 12725-12731. , 20) hshioiierglto fteactin the of regulation Phosphoinositide (2003). ie,A . oksh .M n Ru and M. B. Jockusch, H., A. diger, ¨ 21) hshioiie3kns p110alpha 3-kinase Phosphoinositide (2012). 165 ice.Bohs e.Commun. Res. Biophys. Biochem. 65 1033-1044. , 1047 20) acu ilg ftetransverse the of biology Calcium (2005). 761-789. , 19) iclnkoku eut nheart in results knockout Vinculin (1998). 16 165 icHatFail Heart Circ 20) ada a ucinchannels junction gap Cardiac (2002). 911 o:1047/1/99 doi: 99-111. , 453-460. , ic Res. Circ. 21) elcmuiain nthe in communications Cell (2010). 19) cdcpopoiisinhibit phospholipids Acidic (1996). 371-381. , .Cl Biol. Cell J. Development 21) h-iaeInhibitor, Rho-Kinase (2012). Cell ice.Bohs Res. Biophys. Biochem. elMlNeurobiol. Mol Cell 20) h tutr and structure The (2006). 91 128 104-111. , ic Res. Circ. .Ml el Cardiol. Cell. Mol. J. 5(4) 141 547-560. , o.Cl.Biol. Cell. Mol. 2-3.doi: 523-534. , 755-764. , 20) Vinculin (2005). 125 .Bo.Chem. Biol. J. 327-337. , 20b.A (2006b). 110 349 (2006a). (1998). (2004). diger, ¨ 978- , 709- , Mol. 27 ,

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