fkbp1a controls ventricular myocardium trabeculation and...

RESEARCH ARTICLE1946

Development 140, 1946-1957 (2013) doi:10.1242/dev.089920© 2013. Published by The Company of Biologists Ltd

INTRODUCTIONThe formation of the ventricular walls is essential to cardiogenesisand is required for normal cardiac function (Moorman andChristoffels, 2003). Ventricular trabeculation and compaction areimportant morphogenetic events that are controlled by a precisebalance between cardiomyocyte proliferation and differentiationduring ventricular chamber formation and wall maturation. Thetrabeculation phase of cardiac development is initiated at the endof cardiac looping (E9.0 to E9.5 in mouse) and is defined as thegrowth of primitive cardiomyocytes to form the highly organizedmuscular ridges that are lined by layers of invaginated endocardialcells (Ben-Shachar et al., 1985). The newly formed trabeculaegradually contribute to the formation of the papillary muscles, theinterventricular septum and cardiac conductive cells. Concomitantwith ventricular septation, the trabeculae start to compact at theirbase adjacent to the outer myocardium, adding substantially toventricular wall thickness and coincidently establishing coronarycirculation (Wessels and Sedmera, 2003). A significant reduction in

trabeculation is closely associated with myocardial growth arrest,whereas persistent trabeculation and/or a reduced level ofcompaction are associated with left ventricular noncompaction(LVNC) (Towbin, 2010) [also called left ventricularhypertrabeculation (LVHT)] (Finsterer, 2009). LVNC(MIM300183) is a distinct form of inherited cardiomyopathy(Pignatelli et al., 2003; Sandhu et al., 2008). Although some LVNCpatients survive into adulthood (Weiford et al., 2004), severe casesaccompanied with other cardiac defects (e.g. Ebstein’s anomaly)commonly die at a young age (Koh et al., 2009).

The underlying molecular and cellular mechanisms thatorchestrate ventricular trabeculation and compaction, as well as thepathogenesis of LVNC, are poorly understood due to theheterogeneity of the patients and the lack of suitable genetic models(Srivastava and Olson, 2000). Genetic screening of isolated LVNCpatients has identified a handful of gene mutations in sarcomereproteins (Klaassen et al., 2008; Xing et al., 2006). However, thegenes that are linked to the more severe cases of pediatric LVNChave yet to be identified. Previously, we showed that mutant micedeficient in FK506 binding protein 1a (Fkbp1a, also known asFKBP12) develop multiple abnormal cardiac structures thatphenocopy severe cases of LVNC, including a characteristicincrease in the number and thickness of ventricular trabeculae (i.e.hypertrabeculation), deep intertrabecular recesses, lack ofcompaction that leads to a thin left ventricular wall (i.e.noncompaction) and a prominent ventricular septal defect (VSD)(Shou et al., 1998).

Fkbp1a is a ubiquitously expressed 12 kDa peptidyl-prolylisomerase (Bierer et al., 1990; Schreiber and Crabtree, 1995). Itbinds to FK506 and rapamycin and inhibits calcineurin and mTORactivity. Fkbp1a is associated with multiple intracellular protein

1Riley Heart Research Center, Division of Pediatric Cardiology and 2Division ofNeonatology, Herman B. Wells Center for Pediatric Research, Department ofPediatrics, Indiana University School of Medicine, Indianapolis, IN 46202, USA.3Department of Pediatric Cardiac Surgery, The Second Xiangya Hospital, CentralSouth University, Changsha 410008, China. 4Department of Pharmacology, HarbinMedical University, Harbin 150081, China. 5Department of Biology and Biochemistry,University of Houston, Houston, TX 77204, USA. 6The Heart Institute, Division ofPediatric Cardiology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH45229, USA.

*These authors contributed equally to this work‡Authors for correspondence ([email protected]; [email protected])

Accepted 18 February 2013

SUMMARYTrabeculation and compaction of the embryonic myocardium are morphogenetic events crucial for the formation and function of theventricular walls. Fkbp1a (FKBP12) is a ubiquitously expressed cis-trans peptidyl-prolyl isomerase. Fkbp1a-deficient mice developventricular hypertrabeculation and noncompaction. To determine the physiological function of Fkbp1a in regulating the intercellularand intracellular signaling pathways involved in ventricular trabeculation and compaction, we generated a series of Fkbp1aconditional knockouts. Surprisingly, cardiomyocyte-restricted ablation of Fkbp1a did not give rise to the ventricular developmentaldefect, whereas endothelial cell-restricted ablation of Fkbp1a recapitulated the ventricular hypertrabeculation and noncompactionobserved in Fkbp1a systemically deficient mice, suggesting an important contribution of Fkbp1a within the developing endocardiain regulating the morphogenesis of ventricular trabeculation and compaction. Further analysis demonstrated that Fkbp1a is a novelnegative modulator of activated Notch1. Activated Notch1 (N1ICD) was significantly upregulated in Fkbp1a-ablated endothelial cellsin vivo and in vitro. Overexpression of Fkbp1a significantly reduced the stability of N1ICD and direct inhibition of Notch signalingsignificantly reduced hypertrabeculation in Fkbp1a-deficient mice. Our findings suggest that Fkbp1a-mediated regulation of Notch1plays an important role in intercellular communication between endocardium and myocardium, which is crucial in controlling theformation of the ventricular walls.

KEY WORDS: FK506 binding protein 12, Ventricular hypertrabeculation/noncompaction, Notch1, Endocardial-myocardial signaling, Mouse

Fkbp1a controls ventricular myocardium trabeculation andcompaction by regulating endocardial Notch1 activityHanying Chen1,*,‡, Wenjun Zhang1,*, Xiaoxin Sun1, Momoko Yoshimoto2, Zhuang Chen1, Wuqiang Zhu1, Jijia Liu1,3, Yadan Shen1,3, Weidong Yong1, Deqiang Li1, Jin Zhang1, Yang Lin2, Baiyan Li1,4, Nathan J. VanDusen1, Paige Snider2, Robert J. Schwartz5, Simon J. Conway2, Loren J. Field1, Mervin C. Yoder2,Anthony B. Firulli1, Nadia Carlesso2, Jeffrey A. Towbin6 and Weinian Shou1,‡

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1947RESEARCH ARTICLEFkbp1a in cardiac development

complexes (Ozawa, 2008; Wang and Donahoe, 2004), such asBMP/Activin/TGFβ type I receptors, Ca2+-release channels, andfunctionally regulates the voltage-gated Na+ channel (Maruyama etal., 2011). Previously, we revealed that Bmp10 is upregulated inFkbp1a-deficient hearts. Bmp10-deficient mice die by E10.5 andexhibit hypoplastic and thin ventricular walls and impairedventricular trabeculation (Chen et al., 2004). Transgenicoverexpression of Bmp10 in embryonic heart led to ventricularhypertrabeculation (Pashmforoush et al., 2004). The elevated levelof Bmp10 observed in both Fkbp1a and Nkx2-5 knockout heartscorrelates strongly with the ventricular hypertrabeculation andnoncompaction phenotypes displayed in these mutants (Chen et al.,2009). However, the underlying mechanism by which Fkbp1aregulates Bmp10 expression and ventricular wall formation remainselusive.

Recently, it has been shown that endocardial Notch1 provideskey spatial-temporal control of myocardial growth via regulation ofBmp10 and neuregulin 1 (Nrg1) (Grego-Bessa et al., 2007).Endocardium is primarily made up of endothelial cells. ActivatedNotch1 intracellular domain (N1ICD) was found to be moreabundant in endocardial cells near the proximal end of the trabecularmyocardium, where trabeculation initiates, and was significantlyless abundant in the endocardial cells at the distal end of thetrabeculae (Grego-Bessa et al., 2007). Ablation of Notch1 or itstranscriptional co-factor Rbpjk within endothelial cells results inhypotrabeculation and, subsequently, early embryonic lethality (DelMonte et al., 2007; Grego-Bessa et al., 2007). Interestingly, bothendocardially expressed Nrg1 and myocardially expressed Bmp10were downregulated in endothelial-restricted Notch1 knockouthearts (Grego-Bessa et al., 2007). Collectively, these findingssuggested a crucial role for endocardial Notch1 in regulatingventricular trabeculation.

To determine the cellular and molecular mechanism of Fkbp1a inregulating ventricular trabeculation and compaction, and itspathogenetic role in LVNC, we generated Fkbp1a conditionalknockouts using the Cre-loxP recombination system. AblatingFkbp1a in cardiac progenitor cells via the use of Nkx2.5cre mice(Moses et al., 2001), we were able to generate Fkbp1aflox/–:Nkx2.5cre

mice that recapitulate the ventricular hypertrabeculation andnoncompaction with full penetrance observed in systemic Fkbp1anull mice. By contrast, ablation of Fkbp1a using cardiomyocyte-specific Cre lines did not give rise to abnormal ventricular wallformation. Surprisingly, endothelial-restricted ablation of Fkbp1aphenocopied the ventricular hypertrabeculation and noncompactionobserved in Fkbp1a systemically deficient mice, suggesting thatendocardium plays an important role in regulating ventriculartrabeculation and compaction. Biochemical and molecular analysesdemonstrated that Fkbp1a regulates Notch1-mediated signalingwithin developing endocardial cells. An excess of activated Notch1is found in Fkbp1a-ablated endothelial cells and is the likely causeof the observed Fkbp1a mutant phenotypes. Treatment of Fkbp1a-deficient mice with Notch inhibitors reduces the hypertrabeculationphenotype. Taken together, we have revealed a mechanism wherebyFkbp1a modulates a critical level of Notch1 activity that is requiredfor regulating ventricular chamber and wall formation.

MATERIALS AND METHODSGeneration of Fkbp1a floxed and conditional knockout miceThe generation of Fkbp1a floxed mice (Fkbp1aflox) has been describedpreviously (Maruyama et al., 2011). To achieve cell lineage-restricteddeletion of Fkbp1a in the developing heart, Fkbp1aflox mice were crossed tovarious cell type-specific Cre mouse lines. To ensure efficient Cre-loxP

recombination in these conditional genetic ablations, we first createdFkbp1a+/−/Cre+ mice followed by an additional intercross ontoFkbp1aflox/flox mice. For the most part, we used Fkbp1aflox/–/Cre+ as anexperimental group and Fkbp1aflox/+/Cre+ and Fkbp1aflox/–/Cre– as thecontrol group. Animal protocols were approved by the Indiana UniversitySchool of Medicine Institutional Animal Care and Research AdvisoryCommittee.

Histological, morphological, whole-mount and section in situhybridization, and immunohistochemical analysesEmbryos were harvested by cesarean section. Embryos and isolatedembryonic hearts at specific stages were fixed with 4% paraformaldehydein PBS. The fixed embryos were paraffin embedded, sectioned (7 μm), andstained with Hematoxylin and Eosin. Whole-mount and section in situhybridization were performed as previously described (Franco et al., 2001).In brief, complementary RNA probes for various cardiac markers werelabeled with digoxigenin (DIG)-UTP using the Roche DIG RNA LabelingSystem according to the manufacturer’s guidelines. Immunohistochemicalstaining was performed using the staining system from Vector Laboratoriesaccording to the manufacturer’s instructions. The primary antibodies usedin the immunohistochemical analyses were: anti-Fkbp1a (FKBP12)antibody (Thermo Scientific, PA1-026A), MF-20 anti-myosin heavy chainmonoclonal antibody [Developmental Studies Hybridoma Bank (DSHB),University of Iowa], anti-Ki67 antibody (ab15580; Abcam), anti-CD31(Pecam1) antibody (BD Biosciences, 553370) and anti-cleaved Notch1(N1ICD) antibody (Cell Signaling, 2421s).

Whole-mount immunofluorescence staining and confocalmicroscopy imagingStaining and microscopy procedures were as previously described (Chen etal., 2004). Embryos were harvested and washed three times in ice-cold PBSand fixed for 10 minutes in pre-chilled acetone before being treated withblocking solution containing 3% non-fat dried milk (Bio-Rad) and 0.025%Triton X-100 for 1 hour. Directly conjugated primary antibodies were thenadded to a final concentration of 1 μg/ml for 12-18 hours at 4°C. The anti-CD31 antibody (BD Biosciences) and the MF-20 monoclonal antibody(DSHB) were labeled with Alexa Fluor 488 and the anti-Fkbp1a antibody(Thermo Scientific) was labeled with Alexa Fluor 647 using a monoclonalantibody labeling kit (Molecular Probes). Samples were analyzed using aBio-Rad MRC 1024 laser scanning confocal microscope equipped with akrypton-argon laser (488, 647 nm). z-series were obtained by imaging serialconfocal planes at 512×512 pixel resolution with a Nikon 20× oil-immersion objective (2 μm intervals).

EchocardiographyMice were gently anesthetized with 1.5% isoflurane. Two-dimensionalshort-axis images were obtained with a high-resolution micro-ultrasoundsystem (Vevo 770, VisualSonics) equipped with a 40 MHz mechanical scanprobe. Fractional shortening (FS), ejection fraction, left ventricular internaldiameter (LVID) during systole, LVID during diastole, end-systolic volume,and end-diastolic volume were calculated with Vevo Analysis software(version 2.2.3) as described previously (Zhu et al., 2009). LVID duringsystole and diastole were measured from M-mode recording at the level ofthe mid-papillary muscle, whereas end-systolic and end-diastolic volumeswere measured with B-mode recording in a plane containing the aortic andmitral valves.

Cell transfection and protein stability assayFlag-tagged human FKBP1A cDNA was subcloned into the BamHI andEcoRI sites of the pCDNA3 vector (Invitrogen). N1ICD expression plasmid(N1ICD-PCS2) is a generous gift from Dr Raphael Kopan (WashingtonUniversity). Western blots employed anti-Flag (Sigma, F1804), anti-N1ICD(Cell Signaling) and anti-tubulin (Sigma, T6199) antibodies. For the proteinstability assay, the N1ICD-expressing plasmid was transfected into controlHEK293 cells and those stably expressing human FKBP1A tagged withFlag epitope (FKBP1A-HEK293) using FuGENE HD (Roche) according tothe manufacturer’s instructions. Cycloheximide (50 µg/ml, Sigma) wasadded to the medium for the indicated length of time. To inhibitproteasomes, transfected cells were treated with the proteasome inhibitor D

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MG-132 (10 μmol, EMD Chemicals) for various time periods. For detectionof ubiquitylated N1ICD, histidine-tagged ubiquitin (His-ubiquitin)pCDNA3 plasmid was co-transfected with N1ICD expression plasmid intoHEK293 or FKBP1A-HEK293 cells. After 30 hours, transfected cells wereincubated with MG-132 (10 μmol) for 8 hours before being lysed in Ni-agarose lysis buffer [50 mM NaH2PO4, 300 mM NaCl, 5 mM imidazole,0.05% Tween 20, 100 µg/ml N-ethylmaleimide, and Complete ProteaseInhibitor (Roche)]. His-ubiquitin-conjugated proteins were enriched usingNi-NTA-agarose (Qiagen) from cells co-transfected with N1ICD and His-ubiquitin. The beads were washed ten times with Ni-agarose wash buffer(50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, 0.05% Tween 20). Inorder to reduce nonspecific binding to beads as much as possible, nickel-binding proteins were resuspended in 2× sample buffer supplemented with200 mM imidazole and heated for 10 minutes at 95°C before western blotwith anti-N1ICD antibody.

Luciferase assayHes1-pGL3 luciferase plasmid and full-length Notch1 and Dll4 expressionplasmids have been described previously (McGill and McGlade, 2003).Fkbp1a knockout and control mouse embryonic fibroblasts (MEFs) andendothelial cells were isolated as described (Weaver et al., 1997). For theluciferase assay, Hes1-pGL3 reporter plasmid was transfected into Fkbp1aknockout or control MEFs and endothelial cells alone or with differentcombinations of Notch1, Dll4 and Fkbp1a expression plasmids. Renillaluciferase plasmid was co-transfected as an internal control. Transfectionefficiency was determined by co-transfection of pCDNA3-EGFP. Luciferaseactivity was analyzed 36 hours after transfection with the Dual-LuciferaseAssay System (Promega) according to the manufacturer’s instructions.

Isolation and culture of endothelial cellsMurine endothelial cells were isolated from lung tissues of Fkbp1aflox/flox

neonates (day 10). The harvested lung tissue was minced to small piecesand then digested with 0.25% collagenase A (Stem Cell Technologies) for40 minutes followed by cell dissociation buffer (Life Technologies) in orderto produce a single-cell suspension. The cell suspension was filtered througha 70-μm strainer and stained with anti-CD31, anti-CD45 (Ptprc) and anti-Mac1 (Itgam) antibodies (all eBiosciences). The CD45– Mac1– CD31+

endothelial cells were sorted on a FACS Aria (Becton Dickinson). Thesorted endothelial cells were cultured in EGM2 (endothelial growth medium2, Lonza) with 10% FBS, and 7 days after initiating culture the adherentcells were infected with recombinant retrovirus expressing humantelomerase to maintain proliferation potential (Hahn et al., 1999). After 2weeks of further culture, the endothelial cells were reselected and enrichedfor CD45– Mac1– CD31+ cells by FACS sorting. The resulting endothelialcells were analyzed by FACS and confirmed by immunofluorescencestaining for endothelial cell surface markers including CD31 (eBiosciences),Vcam1 (Santa Cruz) and Flk1 (Kdr) (eBiosciences) (see supplementarymaterial Fig. S3A,B). To further validate the endothelial characteristics ofthe cells, we used an in vitro MatriGel tube-forming assay (seesupplementary material Fig. S3C). As previously described, we platedFkbp1aflox/flox cells on a thin layer of MatriGel (Stem Cell Technologies) at1×104 cells/well of a 96-well plate in ECM2 containing 10% FCS andallowed a tubular structure to form. Typical tube formation after 20 hoursin culture (supplementary material Fig. S3) suggested that the isolatedFkbp1aflox/flox cells maintained endothelial cell characteristics. To generateFkbp1a-deficient endothelial cells, Fkbp1aflox/flox endothelial cells wereinfected with recombinant adenovirus expressing Cre/eGFP (Zhang et al.,2004), in which the eGFP signal was used to evaluate transductionefficiency. Western blot and qRT-PCR analyses were used to confirm theexpression levels of Fkbp1a in these cells. For plasmid transfection ofendothelial cells, X-tremeGENE HP DNA transfection reagents (RocheDiagnostics) were used according to the manufacturer’s instructions.

To test Notch1 protein stability in endothelial cells, N1ICD-PCS2 was co-transfected into Fkbp1aflox/flox endothelial cells with empty pCDNA3plasmid at a 10 to 1 ratio using FuGENE HD (Roche Applied Bioscience)according to the manufacturer’s instructions. Cells stably expressing N1ICDwere selected by neomycin resistance with G418 (Invitrogen, 400 ng/ml inculture medium). N1ICD-expressing Fkbp1a-deficient endothelial cells

were generated by infection with recombinant adenovirus expressingCre/eGFP (1×108 pfu). Cycloheximide (25 µg/ml) was added to the mediumfor the indicated length of time followed by western blot analysis.

γ-Secretase inhibitor injectionThe γ-secretase inhibitor DBZ {(S,S)-2-[2-(3,5-difluorophenyl)acetylamino]-N-(5-methyl-6-oxo-6,7-dihydro-5H-dibenzo[b,d]azepin-7-yl)propionamide} (EMD Chemicals, 565789) was given by intraperitoneal injection at 11.5days of pregnancy (10 μmol/kg) (Milano et al., 2004). Control mice wereadministered with vehicle (0.5% hydroxypropylmethylcellulose in 0.1%Tween 80). Embryos were harvested at E13.5 and subjected tomorphological, histological and in situ hybridization analyses as describedabove.

Statistical analysisAll values are presented as mean ± s.e.m. Differences between groups werecompared by Student’s t-test. P<0.05 was considered significant.

RESULTSCardiac progenitor ablation of Fkbp1aTo determine whether the ventricular hypertrabeculation andnoncompaction present in Fkbp1a-deficient mice is a primarycardiac defect, we used Nkx2.5cre to ablate the Fkbp1a gene.Nkx2.5cre delivers Cre within cardiac progenitor cells in the cardiaccrescent, which give rise to cardiomyocytes, endocardial cells andto a subpopulation of cells from the septum transversum that givesrise to the epicardium, and within cardiac neural crest cells (Moseset al., 2001; Zhou et al., 2008). Fkbp1aflox/–:Nkx2.5cre micedemonstrated fetal lethality, dying in utero between E14.5 and birth.The mutants phenocopied Fkbp1a-deficient mice, with profoundhypertrabeculation, noncompaction and VSDs with 100%penetrance (Fig. 1; Table 1) (Shou et al., 1998), indicating that theventricular hypertrabeculation and noncompaction within Fkbp1asystemic knockouts are primarily derived from defects in thecardiogenic program, and are not secondary to defects in anotherorgan system.

Specific ablation of Fkbp1a within myocardial,epicardial and neural crest cell-restricted cellpopulationsTo further determine the cell-autonomous contribution to theventricular hypertrabeculation and noncompaction caused byFkbp1a ablation, we crossbred Fkbp1aflox mice onto thecardiomyocyte-specific sodium-calcium exchanger promoter H1-cre (H1-Ncx-cre) (Müller et al., 2002). The Cre activity in H1-Ncx-cre is observed in developing myocardium as early as E9.5 and ina cardiomyocyte-restricted manner throughout embryonic, fetal andadult life (supplementary material Fig. S1). To our surprise,Fkbp1aflox/–:H1-Ncx-cre mice demonstrated normal ventricularchamber formation and survived to adulthood. Histologically, nohypertrabeculation or noncompaction was found in Fkbp1aflox/–:H1-Ncx-cre hearts (Fig. 2; Table 1). Western blot, qRT-PCR andimmunofluorescence analyses confirmed efficient ablation ofFkbp1a within the embryonic myocardium (Fig. 2). This findingsuggests that cardiomyocytes are not the primary cell typecontributing to the ventricular hypertrabeculation andnoncompaction observed in mice with systemic Fkbp1a ablation.

To validate this observation, we crossbred Fkbp1flox mice to othercardiomyocyte-specific Cre transgenic lines, including cTnt-cre(Table 1) (Jiao et al., 2003; Wang et al., 2000). Consistently, all ofthese mutant mice exhibited normal ventricular chamber formation.cTnt-cre delivers Cre within the developing myocardium as early asE7.5 (Jiao et al., 2003), which is similar to that reported for Nxk2.5cre. D

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Despite this early Cre activity, Fkbp1aflox/–:cTnt-cre mice displaynormal ventricular trabeculation and compaction (supplementarymaterial Fig. S2), further indicating that the abnormal ventriculardevelopment in Fkbp1a-deficient mice is not a direct function ofFkbp1a within cardiomyocytes. Similarly, cardiac development wasreported to be normal in studies using MHC-cre and Mck-cre to deleteFkbp1a (Maruyama et al., 2011; Tang et al., 2004). Together, thesedata rule out cardiomyocytes as the primary contributing cell lineageto ventricular hypertrabeculation and noncompaction in Fkbp1a-deficient mice, and instead imply another cell lineage or lineages asthe primary cause.

Epicardium has been implicated to play an important role insupporting ventricular wall growth and, potentially, compaction(Sucov et al., 2009). To test whether epicardial Fkbp1a contributesto hypertrabeculation or noncompaction, we ablated Fkbp1a fromepicardial cells using Tbx18cre mice (Cai et al., 2008). Tbx18cre micedeliver Cre activity largely to epicardium, with a low activity incardiomyocytes refined in ventricular septum (Cai et al., 2008;Christoffels et al., 2009). We found that Fkbp1aflox/–:Tbx18cre

mutants had normal ventricular wall formation (Table 1;supplementary material Fig. S2). Previously we showed that, despitelow penetrance, Fkbp1a-deficient mice develop exencephaly (Shouet al., 1998), indicating potential involvement of cranial-facialneural crest cells. We used Wnt1-cre to ablate Fkbp1a from neuralcrest cell lineages (Jiang et al., 2000). Once again, we observednormal ventricular wall formation in Fkbp1aflox/–:Wnt1-cre mutants(Table 1; supplementary material Fig. S2). When considering thecell lineages marked by Nkx2.5cre, the endocardium remaineduntested. Therefore, we next sought to specifically ablate Fkbp1afrom the endocardium.


Endothelial-specific ablation of Fkbp1aThe developing endocardium has an important role in regulatingventricular wall formation (Brutsaert, 2003). To test the contributionof endocardium to the hypertrabeculation and noncompactionphenotypes observed with systemic Fkbp1a ablation, we studied theendothelial-specific deletion of Fkbp1a using Tie2-cre (Tie2 is alsoknown as Tek) transgenic mice (Constien et al., 2001; Gitler et al.,2004; Kisanuki et al., 2001). Tie2-cre mice deliver Cre activity toendothelial cells, including endocardial cells, as early as E9.0(Kisanuki et al., 2001). Fkbp1aflox/–:Tie2-cre mice phenocopied theventricular hypertrabeculation and noncompaction seen in Fkbp1a-deficient hearts (Fig. 3; Table 1). Approximately 35% (15/45) ofE13.5-14.5 Fkbp1aflox/–:Tie2-cre embryos were edematous andappeared to have failing hearts as evidenced by the absence ofeffective perfusion. Hearts isolated from these E13.5-14.5Fkbp1aflox/–:Tie2-cre embryos were enlarged and typically‘pumpkin-shaped’, lacking the normal ventricular groove, a strongindication of abnormal ventricle chamber development (Fig. 3).Histological analysis of these hearts confirmed multiple abnormalcardiac ventricular structures similar to those of Fkbp1a−/− hearts,including various degrees of hypertrabeculation, noncompactionand a thin compact wall. Prominent VSDs, including bothmembranous and muscular, were also observed in six of these 15mutant hearts.

Although the majority of Fkbp1aflox/–:Tie2-cre mice (comparedwith just ~5% of Fkbp1a−/− mice) survived in utero, most of thesesurvivors died within 8 weeks of birth, probably owing tocompromised cardiac function (see below) and defects in T cellfunction (data not shown); the surviving Fkbp1aflox/–:Tie2-cre miceexhibited milder hypertrabeculation and noncompaction phenotypes

Fig. 1. Genetic ablation of Fkbp1a using Nkx2.5cre.(A) Comparison of gross morphology ofFkbp1aflox/–:Nkx2.5cre and control mouse embryos (top)and hearts (bottom) at E14.5. Fkbp1aflox/–:Nkx2.5cre

embryos appear edematous (red arrow) and die inutero. Gray arrow indicates formation of the ventriculargroove in the control heart. (B) Histological analysis ofFkbp1aflox/–:Nkx2.5cre and control hearts at E14.5. Arrowsindicate the overgrowth of the trabecular myocardiumin Fkbp1a mutant hearts. The width of the ventricularcompact wall is indicated. The boxed regions areenlarged beneath. RA, right atrium; LA, left atrium; RV,right ventricle; LV, left ventricle; VSD, ventricular septaldefect.

Table 1. Phenotypes of cell lineage-restricted Fkbp1a mutants

Ablation Cre line Phenotype n (mutant, control, litters)*

Cardiac Nkx2.5cre 100% embryonic lethality with ventricular hypertrabeculation, 10, 12, 7noncompaction and VSD

Cardiomyocyte H1-Ncx-cre 100% viable and fertile, normal ventricular development 11, 35, 12Endothelial Tie2-cre ~35% with ventricular hypertrabeculation, noncompaction and VSD 45, 22, 31 (E13.5 and E14.5)Epicardial Tbx18cre 100% viable and fertile, normal ventricular development 5, 5, 4Neural crest cell Wnt1-cre 100% survive to postnatal, normal ventricular wall development, but 100% 25, 25, 17

die within 1 month of birth due to defect in thymus development

*The number of mutant and control embryos analyzed from the indicated number of litters, respectively.VSD, ventricular septal defect. D

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(Fig. 4). Echocardiographic analysis demonstrated severelycompromised cardiac function in these mutants, consistent with thehistological findings (Fig. 4). Collectively, our observations indicatethat abnormal endocardial function is likely to be the primary causeof the hypertrabeculation and noncompaction phenotypes inFkbp1a-deficient mutants.

Notch1 and downstream endocardial signaling arealtered in Fkbp1a mutant heartsRecent studies have demonstrated that perturbation of Notch1signaling within the endocardium impairs cardiomyocyte Bmp10expression, ventricular trabeculation and wall formation (Grego-Bessa et al., 2007). To define whether Notch1 activation within theendocardium is altered in Fkbp1a mutants, we compared the patternof activated N1ICD in the developing ventricular endocardial cells

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of Fkbp1a-deficient, Fkbp1aflox/–:Tie2-cre and wild-type controlhearts. Using immunohistochemistry, we were able to confirm thatnuclear N1ICD was robustly distributed within the endocardial cellsclose to the proximal end of the trabecular myocardium of controlhearts (Fig. 5A). By contrast, this characteristic pattern of N1ICDwas clearly altered in Fkbp1a mutant hearts, as N1ICD was foundin endocardial cells throughout and alongside the trabecularmyocardium (Fig. 5A), suggesting that Notch1 is activatedectopically within Fkbp1a-deficient and Fkbp1aflox/–:Tie2-creendocardium.

Furthermore, Hey1 and ephrin B2 (Efnb2), two well-knowndirect downstream targets of Notch1 that are expressed withinendocardium (Grego-Bessa et al., 2007; Hainaud et al., 2006),were found significantly upregulated in the Fkbp1a-deficientendocardial cells, supporting the observation that there isincreased Notch1 activity in Fkbp1a mutant endocardium(Fig. 5B,C). Interestingly, although the overall level of Hey2expression did not appear to differ significantly in Fkbp1a mutantversus control heart (Fig. 5C), its expression pattern wassignificantly altered (Fig. 5B). In normal myocardium, Hey2transcript is restricted to compact myocardium. We found thatHey2 expression is expanded into the trabecular myocardium inFkbp1a mutant heart (Fig. 5B). Endocardial Nrg1 expression isdownstream of Efnb2 and is downregulated in Notch1 mutantendocardium (Grego-Bessa et al., 2007). Both in situ hybridizationand qRT-PCR analyses confirmed that Nrg1 was upregulated inFkbp1aflox/–:Tie2-cre heart, as was Bmp10 (Fig. 5B,C). Takentogether, our data demonstrate that Fkbp1a is an importantregulator for endocardial Notch1-mediated signaling duringventricular wall formation.

Fkbp1a regulates N1ICD stability and turnoverTo further define the role of Fkbp1a in Notch1 activation, weisolated mouse embryonic fibroblasts (MEFs) from Fkbp1a-deficient and control embryos (E12.5) and analyzed Notchactivity. Notch-dependent Hes1-luciferase reporter plasmid wastransfected into Fkbp1a-deficient and wild-type control MEFs incombination with Notch1 and delta-like 4 (Dll4) expressionplasmids. The level of luciferase activity reflects the level ofactivated Notch1 (McGill and McGlade, 2003). From fourindependent sets of experiments, we found that luciferaseactivities were significantly enhanced in both Dll4-transfected andDll4/Notch1 co-transfected Fkbp1a-deficient MEFs whencompared with transfected wild-type MEFs (Fig. 6A). Re-introduction of Fkbp1a into the Fkbp1a-deficient cells reducedluciferase activity to a level comparable to that in wild-type cells.Consistent with these findings, western blot analysis demonstratedthat the endogenous N1ICD level is significantly higher inFkbp1a-deficient than in wild-type MEFs (Fig. 6A). To validatethis finding in endothelial cells, we prepared endothelial cells fromFkbp1aflox/flox neonatal mice (supplementary material Fig. S3).Fkbp1a-deficient endothelial cells were generated by transducingthe cells with Ad-Cre/eGFP. Western blot confirmed the efficientablation of Fkbp1a in Cre-transduced Fkbp1aflox/flox mouseendothelial cells (Fkbp1aflox/flox/Cre) (Fig. 6B). Hes1-luciferasereporter assays were performed in Fkbp1aflox/flox andFkbp1aflox/flox/Cre cells (Fig. 6B). Similar to the finding in MEFs,Notch-dependent luciferase activities were significantly higher inFkbp1aflox/flox/Cre than in Fkbp1aflox/flox endothelial cells (Fig. 6B).

We transfected activated Notch1 (N1ICD-PCS2) into Fkbp1aflox/flox

endothelial cells in conjunction with pCDNA3-FKBP1A (human) orcontrol pCDNA3-eGFP vector. The N1ICD protein level in

Fig. 2. Cardiomyocyte-restricted ablation of Fkbp1a using H1-Ncx-cre transgenic mice. (A) Dual immunofluorescence analyses confirm thegenetic ablation of Fkbp1a in developing myocardium at E12.5.Representative confocal images of heart sections co-stained with AlexaFluor 647-conjugated anti-Fkbp1a antibody (red) and Alexa Fluor 488-conjugated anti-myosin heavy chain antibody (MF-20; green). There is asignificant reduction in Fkbp1a in Fkbp1aflox/–:H1-Ncx-cre myocardium.The boxed regions of the merge are enlarged to the right. (B) Westernblot confirms the efficient removal of Fkbp1a in Fkbp1aflox/–:H1-Ncx-creheart. (C) Morphological and histological analysis of Fkbp1aflox/–:H1-Ncx-cre and control hearts at E14.5. RA, right atrium; LA, left atrium; RV, rightventricle; LV, left ventricle.

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Fkbp1aflox/flox FKBP1A-overexpressing endothelial cells wassignificantly lower than in Fkbp1aflox/flox eGFP-expressing cells(Fig. 6C, top). As expected, overexpression of FKBP1A reducedNotch-dependent Hes1-luciferase activity (Fig. 6C, bottom).

To determine whether Fkbp1a could affect endogenous Notch1expression in endothelial cells, we compared Notch1 mRNA levelsin Fkbp1a-deficient and human FKBP1A-overexpressingendothelial cells (Fig. 6D). Notch1 mRNA levels were not alteredin Fkbp1a-deficient or FKBP1A-overexpressing endothelial cells.However, the mRNA levels of the Notch1 downstream targetsEfnb2, Hey1 and Hey2 were upregulated in Fkbp1a-deficient anddownregulated in FKBP1A-overexpressing endothelial cells,consistent with the notion that Notch1 activities are impacteddirectly by the intracellular levels of Fkbp1a.

Previously, we established HEK293 cell lines withoverexpression of Flag-tagged human FKBP1A (FKBP1A-HEK293). When we attempted to overexpress N1ICD in HEK293and FKBP1A-HEK293 cells, we found that the N1ICD protein levelwas significantly reduced in FKBP1A-HEK293 cells whencompared with control HEK293 cells (supplementary materialFig. S4). Given that Notch activity is closely associated withubiquitylation-mediated degradation, this finding, along with theobservation that there is no change in Notch1 mRNA levels and thatN1ICD protein is significantly higher in Fkbp1a-deficient cells,suggested that, mechanistically, Fkbp1a regulates Notch1 activityby modulating N1ICD stability. To test this hypothesis, wecompared the N1ICD protein degradation rate in human FKBP1A-overexpressing (i.e. FKBP1A-HEK293) and control HEK293 cells.


The rate of N1ICD degradation was significantly greater inFKBP1A-HEK293 cells (Fig. 7Aa,b). To validate this finding inendothelial cells, we generated stable N1ICD-overexpressingFkbp1aflox/flox cells (N1ICD-Fkbp1aflox/flox) and compared theirN1ICD degradation rate with that of N1ICD-Fkbp1aflox/flox/Crecells. N1ICD-Fkbp1aflox/flox/Cre cells had a significantly slowerdegradation rate than that of N1ICD-Fkbp1aflox/flox cells(Fig. 7Ac,d).

We also evaluated the rate of N1ICD translation. There is nonoticeable difference in N1ICD protein synthesis betweenFKBP1A-HEK293 cells and controls (Fig. 7B; supplementarymaterial Fig. S5). We next blocked ubiquitylation-mediated proteindegradation using the proteasome inhibitor MG-132, and assessedthe amount of ubiquitylated N1ICD in FKBP1A-HEK293 andcontrol cells. The amount of ubiquitylated N1ICD was significantlyhigher in FKBP1A-HEK293 cells than in HEK293 control cells(Fig. 7C). Taken together, these data indicate that Fkbp1a regulatesthe level of activated Notch1 by controlling its stability viaubiquitin-mediated protein turnover.

The γ-secretase inhibitor DBZ reduceshypertrabeculation in Fkbp1a-deficient heartsClearly, elevated Notch signaling is directly associated withabnormal trabeculation in Fkbp1a mutant embryos. We thereforereasoned that reducing Notch1 activation in vivo would be able torescue the ventricular morphological defects in Fkbp1a mutanthearts, thereby validating the hypothesized mechanism. We appliedthe γ-secretase inhibitor DBZ (Milano et al., 2004) to Fkbp1a

Fig. 3. Endothelial-restricted ablation of Fkbp1a usingTie2-cre transgenic mice. (A) Dual immunofluorescence andPCR analyses to confirm the genetic ablation of Fkbp1a indeveloping endocardial/endothelial cells at E12.5.Representative confocal images of heart sections are co-stained with Alexa Fluor 647-conjugated anti-Fkbp1aantibody (red) and Alexa Fluor 488-conjugated anti-CD31antibody (green). There is a significant reduction of Fkbp1a inthe CD31-positive endothelial cells in Fkbp1aflox/–:Tie2-crehearts. Beneath is shown a diagnostic genomic PCR analysisof DNA samples isolated from E13.5 heart alongside aschematic of the Fkbp1aflox allele showing the location ofdiagnostic primers p1, p2 and p3; E, exon. A specific p3-p2band represents the allele after Cre-loxP-mediatedrecombination. (B) Morphological and histological analysis ofFkbp1aflox/–:Tie2-cre and control embryos and hearts at E14.5.Fkbp1aflox/–:Tie2-cre embryos are edematous (red arrow). Themutant hearts lack a normal ventricular groove (yellowarrows) and demonstrate hypertrabeculation andnoncompaction (black arrows). RA, right atrium; LA, leftatrium; RV, right ventricle; LV, left ventricle; VSD, ventricularseptal defect.

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mutant embryos at E11.5, a stage prior to the appearance of thehypertrabeculation and noncompaction phenotypes (Chen et al.,2009). The pregnant Fkbp1a+/− females that were bred withFkbp1a+/− males were administrated with DBZ (10 μM/kg viaintraperitoneal injection, n=6 females) and vehicle (100 μl, n=6females). The embryos were harvested at E13.5 and processed formorphological and histological examination. Results show that


administration of DBZ significantly diminished thehypertrabeculation phenotypes in Fkbp1a-deficient hearts whencompared with vehicle-treated controls (Fig. 8A,Ba). The ratio oftrabeculae thickness to compact wall thickness approached a normalmorphological distribution (Fig. 8Bc). Remarkably, ventricularseptum formation was also greatly improved in all DBZ-treatedFkbp1a-deficient hearts.

Fig. 4. Histological and functional analyses of adultFkbp1aflox/–:Tie2-cre mice. (A) (Left) Restricted growth of survivingFkbp1aflox/–:Tie2-cre mice. (Right) Abnormal ventricular wallstructure in Fkbp1aflox/–:Tie2-cre hearts, with hypertrabeculation andnoncompaction. Arrows indicate abnormal trabecular myocardiaand ventricular septum. (B) M-mode echocardiographic analysis of2-month-old adult males. One-second traces are shown. Bothfractional shortening and ejection fraction are severelycompromised in Fkbp1aflox/–:Tie2-cre mutants. Values indicate mean± s.e.m.; n=12; *P<0.05. LVIDd, left ventricular internal diameterdiastolic; LVIDs, left ventricular internal diameter systolic; FS%,fractional shortening; LV vol d, left ventricular volume diastolic; LVvol s, left ventricular volume systolic; EF%, ejection fraction; HR,heart rate.

Fig. 5. Assessment of Notch1-mediated signaling in thedeveloping endocardium of Fkbp1a-deficient andFkbp1aflox/–:Tie2-cre mouse hearts. (A) Immunohistologicalanalysis using antibody specific to activated Notch1 (N1ICD). Incontrol heart (WT), nuclear N1ICD is mainly located inendothelial cells at the proximal end of trabeculae (arrows in a�).By contrast, N1ICD was found throughout endothelial cells inboth Fkbp1a-deficient and Fkbp1aflox/–:Tie2-cre mutant hearts(arrows in b� and c�). The boxed regions in a-c are magnified ina�-c�. (B) In situ hybridization of downstream targets of Notch1 inFkbp1aflox/–:Tie2-cre and control hearts at E12.5. Ephrin B2 (Efnb2),neuregulin 1 (Nrg1) and Hey1 are upregulated in endocardialcells in Fkbp1aflox/–:Tie2-cre hearts. Bmp10 is upregulated in bothtrabecular and compact myocardium. Interestingly, Hey2expression is upregulated in trabecular myocardium comparedwith controls. Arrows indicate positive signals. (C) qRT-PCRconfirms the expression levels of Notch1, Efnb2, Nrg1, Hey1, Hey2and Bmp10 in Fkbp1aflox/–:Tie2-cre hearts at E13.5. Interestingly,despite the altered expression pattern, the overall expressionlevel of Hey2 is not altered. Error bars indicate s.e.m. LV, leftventricle.

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Consistent with the histological findings, in situ hybridizationdemonstrated that the levels of endocardial Efnb2 and Nrg1, as wellas myocardial Bmp10, were significantly reduced in DBZ-treatedas compared with vehicle-treated Fkbp1a−/− hearts (Fig. 8C). Asfurther validation, Ki67 immunohistological staining showed thatDBZ treatment resulted in strong suppression of cardiomyocyteproliferative activities in both trabecular and compact myocardiumwithin Fkbp1a−/− hearts, showing a more wild-type growth profile(Fig. 8C; supplementary material Fig. S6). However, despitesignificant reductions in cardiomyocyte hyperproliferation and themyocardial hypertrabeculation phenotype of DBZ-treated mutanthearts, the thickness of the compact wall (ventricular compaction)did not seem to show significant improvement upon DBZ treatment,suggesting that a Notch-independent signaling pathway mightcontribute in part to the regulation of ventricular wall compaction.Taken together, these findings define an important role for Fkbp1ain regulating Notch1-mediated signaling during ventricular walldevelopment.


DISCUSSIONVentricular hypertrabeculation and noncompaction are congenitalmyocardial anomalies that result from disrupted development of theventricular wall and have been increasingly recognized in the clinic(Chen et al., 2009). Genetic analysis of patients, mostly with isolatedforms of ventricular noncompaction without CHDs (congenital heartdefects), has demonstrated a broad genetic heterogeneity, suggestinga complex and/or multifactorial network contributing to the etiologyand pathogenesis (Chen et al., 2009). Currently, surviving patientscarrying FKBP1A mutations have not been reported. Given thepivotal role of Fkbp1a in ventricular wall formation and theembryonic lethal phenotype of Fkbp1a-deficient mice, patients witha germline FKBP1A loss-of-function mutation would seem to havelittle chance of survival in utero. However, mouse geneticmanipulation provides an excellent opportunity to determine the causeand the pathogenetic network that contributes to the ventricularhypertrabeculation and noncompaction, allowing for the identificationof genetic pathways that regulate normal ventricular development.

Fig. 6. Biochemical analyses of altered Notch1 activity in Fkbp1a mutant cells. (A) (Left) Luciferase assay to determine Notch1 activity in Fkbp1a-deficient and control mouse embryonic fibroblasts (MEFs) in which Hes1-luciferase reporter was transfected together with Notch1, Dll4, Fkbp1a andeGFP as indicated. Fkbp1a-deficient cells maintain significantly higher luciferase activity, which is reduced by Fkbp1a re-introduction. (Right) Westernblot analysis of endogenous N1ICD levels in Fkbp1a-deficient and wild-type MEFs. (B) (Left) Luciferase assay to determine Notch1 activity in Fkbp1a-deficient and control mouse endothelial cells. (Right) Western blot shows that the N1ICD protein level is significantly higher in Fkbp1a-deficient cells. (C) (Top) Western blot showing that the N1ICD protein level is downregulated in mouse endothelial cells overexpressing human FKBP1A. (Bottom)Luciferase assay to determine Notch1 activity in FKBP1A-overexpressing and control endothelial cells. (D) qRT-PCR analysis shows that Notch1 mRNAlevels are not altered in Fkbp1a-deficient or FKBP1A-overexpressing endothelial cells. However, Efnb2, Hey1 and Hey2 expression levels are altered. Errorbars indicate s.e.m.

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As an isomerase, Fkbp1a presumably functions as a chaperonein maintaining the appropriate conformation of its substrateproteins/peptides, which could be relevant to the regulation oftheir functional state and/or stability (Lu et al., 2007). Our findingssuggest that Fkbp1a has a role in regulating endocardial Notch1activity, which is crucial to ventricular wall formation. Ourfindings are entirely consistent with recent reports by Mysliwiecand colleagues that upregulation of Notch1 in endocardial cellsleads to ventricular hypertrabeculation and noncompaction(Mysliwiec et al., 2011; Mysliwiec et al., 2012). Most recently,Yang and colleagues demonstrated that upregulated Notch2activity in Numb/Numb-like compound deficient mutantscontributes to Bmp10 upregulation and ventricularhypertrabeculation and noncompaction phenotypes (Yang et al.,2012), further validating our conclusion. However, it should benoted that Tie2-Cre is not endocardial specific, but targets to allendothelial cells including coronary endothelial cells. Nfatc1-Creexpression is initially restricted to the endocardium, andsubsequently marks cardiac vascular endothelial cells as thecoronary vasculature system develops (Wu et al., 2012). Owingto the spatial-temporal pattern of Cre activity within these lines,we currently cannot distinguish the relative contribution of thedifferent cardiac endothelial cell subtypes. Despite this limitation,it is nonetheless clear that Fkbp1a signaling in cardiac endothelialcells plays a major role in regulating ventricular trabeculation andcompaction.

The biochemical mechanism by which Fkbp1a regulatesNotch1 activity is likely to involve controlling the stability ofN1ICD. Indeed, biochemical analyses in endothelial cells, MEFsand HEK293 cells indicate that Fkbp1a can modulate the stabilityof activated Notch1. It is well-known that Notch signaling isregulated by post-translational modification events. ActivatedN1ICD is cleared and thereby inactivated within the cell byubiquitylation and subsequent proteolysis via the proteasome. This


proteasome-dependent degradation of N1ICD is thought to bemediated by interactions of its PEST (proline, glutamic acid,serine and threonine) domain and a specific E3 ubiquitin ligase,Fbxw7 (Oberg et al., 2001). As a known cis-trans peptidyl-prolylisomerase, one attractive hypothesis is that Fkbp1a regulates theconformation of N1ICD via the proline residues contained withinits PEST domain. To test this hypothesis, we used co-immunoprecipitation to determine whether Fkbp1a is able todirectly bind N1ICD. However, we have not been able toconsistently detect a direct molecular interaction between N1ICDand Fkbp1a (data not shown). This negative result could be due totechnical difficulties in capturing transient hit-and-run typeinteractions between Fkbp1a and N1ICD. Further studyemploying systematic biochemical analyses will be necessary todetermine whether Fkbp1a directly interacts with N1ICD tofacilitate its ubiquitylation, or operates via an indirect mechanism.In addition, as Fkbp1a interacts with several other proteincomplexes, our findings do not exclude the potential involvementof other signaling pathways in contributing to the ventricular walldefects in Fkbp1a mutant hearts. Indeed, although the Notchinhibitor DBZ profoundly reduced cellular hyperproliferation andhypertrabeculation, the noncompaction phenotype was barelyaffected by DBZ (Fig. 8). This could simply reflect the complexdynamics of ventricular compaction, which involves multipleNotch-mediated signaling pathways in multiple cardiac celllineages, or there could be an additional unknown parallelsignaling pathway(s) that synergistically controls ventricularcompaction.

Endothelial cell-restricted ablation of Notch1 and Rbpjkdownregulates the expression of both Efnb2 and Nrg1 withinendothelial cells and Bmp10 within cardiomyocytes (Grego-Bessaet al., 2007). Both Bmp10 and Nrg1 have been suggested to havea major functional role in the spatial-temporal control ofcardiomyocyte proliferation and ventricular trabeculation (Grego-

Fig. 7. Biochemical analysis of N1ICD stability anddegradation. (A) Assessment of the N1ICD degradation ratein HEK293 versus FKBP1A-HEK293 cells and in N1ICD-Fkbp1aflox/flox versus N1ICD-Fkbp1aflox/flox/Cre mouseendothelial cells. Cells were treated with the indicatedconcentrations of cycloheximide (CHX). Representativewestern blots show the N1ICD protein level at different timepoints after CHX treatment in HEK293 cells and endothelialcells (EC) of different genotype (a and c), and representativeanalyses of N1ICD degradation in FKBP1A-HEK293 cellsversus control HEK293 cells (b) and N1ICD-Fkbp1aflox/flox

versus N1ICD-Fkbp1aflox/flox/Cre endothelial cells (d). (B) Representative western blot of three independentexperiments showing that N1ICD protein synthesis is notaffected in FKBP1A-HEK293 cells. (C) Ubiquitylated N1ICD issignificantly more abundant in FKBP1A-HEK293 cells. Todetect the ubiquitylated N1ICD protein, 6× histidine-taggedubiquitin (His-ubiquitin) plasmid was co-transfected withN1ICD expression plasmid into HEK293 or FKBP1A-HEK293cells. Transfected cells were incubated with the proteasomeinhibitor MG-132 (10 μmol) for 8 hours before cell lysis andNi-NTA pulldown assay. Error bars indicate s.e.m.

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Bessa et al., 2007; Shi et al., 2003). Interestingly, Bmp10expression can be specifically upregulated by Nrg1 (Kang andSucov, 2005). In addition, Nrg1 hypomorphic mutants have asignificantly reduced Bmp10 expression level (Lai et al., 2010).These findings, both in vitro and in vivo, suggest the interestingidea that Bmp10 is likely to be a downstream target of Nrg1.Endocardial-derived Nrg1 is of particular interest as it signalsthrough its receptor complex, ErbB2/4, which is present only inadjacent cardiomyocytes (Lemmens et al., 2006), providing aunique mechanism for intercellular interactions between twodifferent cardiac cell populations. In zebrafish, the heart is highlytrabeculated. A recent study using zebrafish heart as a modelsystem demonstrated that a Nrg1-ErbB2/4 signaling cascade hasa dual function: in addition to promoting cardiomyocyteproliferation, it regulates cardiomyocyte delamination andmigration (Liu et al., 2010). This dual activity ultimately controlstrabeculation in zebrafish. Interestingly, analysis of endocardial-restricted Rbpjk knockouts has shown that exogenous Bmp10rescues the observed myocardial proliferative defect, whereasNrg1 does not (Grego-Bessa et al., 2007). Apparently, the Nrg1-ErbB cascade is likely to be part of the evolutionarily conservedmechanism for the regulation of cardiomyocyte delamination thatallows for ventricular trabeculation (Liu et al., 2010) or for theregulation of cardiomyocyte differentiation via its pro-differentiation activity (Lai et al., 2010). It would be interesting todetermine whether Bmp10 has a similar function in regulating


cardiomyocyte proliferation and trabeculation in the highlytrabeculated zebrafish heart.

In summary, our study demonstrates a novel genetic andbiochemical role for Fkbp1a in regulating Notch1 activity withinthe endocardium that in part facilitates the intercellular interplaybetween endocardium and myocardium necessary to define thenormal levels of ventricular trabeculation and compaction in themammalian heart.

AcknowledgementsWe thank Dr Shaoliang Jing and William Carter of Indiana University mousecore for their expert assistance.

FundingThis work was supported in part by the National Institutes of Health [grantsHL81092 to W.S. and HL85098 to L.J.F., A.B.F., S.J.C. and W.S.]; and by RileyChildren’s foundation (to L.J.F., A.B.F., S.J.C. and W.S.). Deposited in PMC forrelease after 12 months.

Competing interests statementThe authors declare no competing financial interests.

Supplementary materialSupplementary material available online athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.089920/-/DC1

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