Inhibition of Notch1-Dependent Cardiomyogenesis Leads to a Dilated Myopathy in the Neonatal Heart

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InhibitionofNotch1-DependentCardiomyogenesisLeadstoaDilatedMyopathyintheNeonatalHeart

ARTICLEinCIRCULATIONRESEARCH·AUGUST2010

ImpactFactor:11.02·DOI:10.1161/CIRCRESAHA.110.218487·Source:PubMed

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Inhibition of Notch1-Dependent Cardiomyogenesis Leads to aDilated Myopathy in the Neonatal Heart

Konrad Urbanek, Mauricio Castro Cabral-da-Silva, Noriko Ide-Iwata, Silvia Maestroni,Francesca Delucchi, Hanqiao Zheng, João Ferreira-Martins, Barbara Ogórek, DomenicoD’Amario, Michael Bauer, Gianpaolo Zerbini, Marcello Rota, Toru Hosoda, Ronglih Liao,Piero Anversa, Jan Kajstura, and Annarosa LeriDepartments of Anesthesia and Medicine, and Cardiovascular Division, Brigham and Women’sHospital, Harvard Medical School, Boston, MA 02115

AbstractRationale—Physiological hypertrophy in the developing heart has been considered the productof an increase in volume of preexisting fetal cardiomyocytes in the absence of myocyte formation.

Objective—In this study, we tested whether the mouse heart possesses at birth a pool of cardiacstem cells (CSCs) which differentiate into myocytes contributing to the expansion of theparenchymal cell compartment postnatally.

Methods and Results—We have found that the newborn heart contains a population of c-kit-positive CSCs which are lineage negative, self-renewing and multipotent. CSCs express theNotch1 receptor and show the nuclear localization of its active fragment, N1ICD. In 60% of cases,N1ICD was coupled with the presence of Nkx2.5 indicating that the commitment of CSCs to themyocyte lineage is regulated by Notch1. Importantly, overexpression of N1ICD in neonatal CSCsexpanded significantly the proportion of transit amplifying myocytes. To establish whether thesein vitro findings had a functional counterpart in vivo, the Notch pathway was blocked in newbornmice by administration of a γ-secretase inhibitor. This intervention resulted in the development ofa dilated myopathy and high mortality. Ventricular decompensation was characterized by a 62%reduction in amplifying myocytes which resulted in a 54% decrease in myocyte number.Following cessation of Notch blockade and recovery of myocyte regeneration, cardiac anatomyand function were largely restored.

Conclusions—Notch1 signaling is a critical determinant of CSC growth and differentiation andwhen this cascade of events is altered cardiomyogenesis is impaired, physiological cardiachypertrophy is prevented and a life threatening myopathy supervenes.

KeywordsCardiac stem cells; Notch1; inhibition of myocyte growth

At birth, the myocardium has to accommodate the abrupt changes in the patterns of bloodflow and circulatory resistance and the increasing demands of the rapidly growing organism.Physiological hypertrophy in the developing heart has been considered the result of anincrease in volume of preexisting fetal cardiomyocytes while the contribution of newmyocytes to the expansion in ventricular mass postnatally was viewed as negligible at best.1

Correspondence: Annarosa Leri, MD, Departments of Anesthesia and Medicine, and Cardiovascular Division, Brigham and Women’sHospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115; [email protected].

NIH Public AccessAuthor ManuscriptCirc Res. Author manuscript; available in PMC 2011 August 6.

Published in final edited form as:Circ Res. 2010 August 6; 107(3): 429–441. doi:10.1161/CIRCRESAHA.110.218487.

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However, the documentation of high levels of DNA synthesis in myocytes together withapoptotic cell death2 indicate that the postnatal maturation of the heart is a complex processin which cardiomyogenesis may play a critical role in the acquisition of the adult heartphenotype.

By viral tagging and clonal marking, we have shown that cardiomyogenesis in the adultmouse heart depends on the activation and differentiation of a pool of cardiac stem cells(CSCs) which express the c-kit antigen.3 Based on these findings, the question can be raisedwhether primitive cells with similar properties are present in the neonatal heart and thegeneration of new myocytes is dictated by lineage commitment of these cells. According tothe traditional model of growth in self-renewing organs, stem cells give rise to progenitor-precursor cells, which then become highly dividing amplifying cells that eventually reachterminal differentiation and growth arrest.4 This hierarchically structured mechanism ofparenchymal cell generation may account for the expansion in myocyte number and volumeof the neonatal heart. CSC-derived amplifying myocytes replicate and concurrentlydifferentiate, mimicking cellular hyperplasia and hypertrophy.5

The possibility exists that the formation of new myocytes mediated by CSC growth is not aminor phenomenon that undergoes sudden downregulation at birth, but it represents acontinuous process that conditions the acquisition of the adult heart phenotype structurallyand functionally. Activation of the Notch1 receptor is the critical determinant of thetransition of CSCs to the compartment of amplifying myocytes and inhibition of thispathway has dramatic negative consequences on the adaptation of the heart to ischemicmyocardial injury.5 Collectively, these observations suggest that a similar mechanism maybe operative in the developing heart and Notch1 signaling modulates CSC differentiationand the formation of functionally-competent myocytes.

MethodsThe number of c-kit-positive CSCs and the number and volume of myocytes together withcapillary density were evaluated quantitatively in neonatal mice in the absence and presenceof γ-secretase inhibition which blocks Notch receptor function. Protocols are described athttp://circres.ahajournals.org.

ResultsCSCs in the Neonatal Heart

The neonatal mouse heart at 3–7 days after birth was characterized by closely packedcardiomyocytes and capillary profiles (Online Figure I). CSCs were clustered in atrial andventricular niches and scattered throughout the myocardium (Figure 1A through 1D; OnlineFigure I). Some of these cells were positive for the myocyte transcription factors Nkx2.5 andMEF2C, or contained the contractile proteins α-sarcomeric actin (α-SA) and α-cardiacactinin. They represented various stages of CSC differentiation (Figure 1A through 1D;Online Figure I). The expression of c-kit in combination with nuclear and cytoplasmicmarkers documented a lineage relationship between CSCs and myocyte formation.Replicating myocytes were negative for c-kit and positive for Ki67 and phospho-H3.Cytokinesis was detected by the expression of Aurora B kinase6 which phosphorylateshistone H3 and is required for cell division (Figure 1E and 1F; Figure 2). Importantly,83±12% of Ki67-positive myocytes expressed Nkx2.5 (Online Figure I).

C-kit-positive CSCs were isolated, expanded and characterized by FACS (Online Figure II).CSCs were negative for transcription factors and cytoplasmic proteins specific of myocytes(GATA4, Nkx2.5, MEF2C, α-SA), endothelial cells (ECs: Ets1, flk1, von Willebrand factor)

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and smooth muscle cells (SMCs: GATA6, α-SMA). They were also negative for markers ofhematopoietic cells (CD34, CD45, prominin/CD133). The absence of CD45 excluded thecontribution of mast cells and the lack of CD34, CD45, prominin and flk1 excluded bonemarrow derived endothelial progenitor cells (EPCs). Similarly, coronary vascular progenitorcells are flk1-positive7 and did not contribute significantly to the CSC pool. Lineagenegative CSCs exposed to differentiating medium lost c-kit and gave rise to myocytes, ECsand SMCs; myocytes constituted the predominant pool. The commitment of CSCs tocardiomyocytes in vitro involved the expression of the same transcription factors (Nkx2.5,MEF2C) and contractile proteins (α-SA) identified in vivo (Online Figure III), suggestingthat the acquisition of the myocyte phenotype in vitro recapitulated the in vivodifferentiation program. Some forming myocytes acquired the electrical characteristics ofventricular cells with fast Na+ current or displayed sino-atrial nodal-like properties withspontaneous action potentials (Figure 3A and 3B). Thus, the developing mouse heartpossesses CSCs which are self-renewing and multipotent.

Notch1 Receptor Function in Neonatal CSCsEarly after birth, 58±18% of CSCs expressed the Notch1 receptor (Figure 3C and 3D);Notch2–4 receptors were detected in significantly smaller fractions of CSCs (Online FigureIV). The membrane bound Notch1 ligand, Jagged1, was distributed on the sarcolemma ofcardiomyocytes and was always found at the interface between these cells and CSCs (Figure3E and 3F; Online Figure IV). The interaction of Jagged1 with the extracellular domain ofNotch1 resulted in the nuclear localization of the active cleaved fragment of the receptor(Figure 3E and 3F), the Notch1 intracellular domain (N1ICD). In contrast, the Delta-like4ligand was distributed predominantly in coronary vessels.5

In 60% of cases, the presence of N1ICD in CSCs was associated with the nuclear expressionof the early myocyte transcription factor Nkx2.5 (Online Figure IV), raising the possibilitythat Notch1 favors the commitment of CSCs to the myocyte lineage. A causal relationshipbetween N1ICD and the transactivation of Nkx2.5 in CSCs has previously been shown inthe adult infarcted mouse heart.5 Additionally, the presence of c-kit alone or Nkx2.5 did notpreclude cell replication (Online Figure V).

To establish whether Notch1 activation in neonatal CSCs plays a critical role in theformation of myocytes, CSCs harvested from newborn mice were transduced with alentivirus carrying N1ICD and characterized 5 and 10 days later (Online Figure VI).Potential target genes of Notch1 were evaluated by quantitative RT-PCR. With respect tocontrol cells infected with a lentivirus carrying EGFP only, N1ICD overexpressionupregulated the downstream effectors of the Notch pathway Hes1, Hey1 and Hey2 togetherwith the target gene Nkx2.5 (Figure 4A; Online Figure VI). Transcripts of SRF remainedrelatively constant. Myocardin mRNA increased while GATA4 decreased. The SMC andEC markers GATA6, Vezf1 and PECAM were downregulated. The transcriptionalrepression of GATA4 and GATA6 is consistent with the presence of several binding sitesfor Hes1 and Hey1 in the promoter region of all members of the GATA family.8 Theenhanced expression of myocardin paralleled the upregulation of Nkx2.5 which is apowerful transactivator of the myocardin promoter.9 The physical interaction of myocardinwith the DNA binding domain of GATA4 has a synergistic effect on the Nkx2.5 enhancerdomain favoring myocyte hypertrophy.10 This cellular response may require thedownregulation of Notch1 signaling which is involved in the early commitment of CSCs andthe formation of cycling immature myocytes.

At 5 days, lentiviral infection with N1ICD resulted in a 3.4-fold increase in the number ofCSCs expressing Nkx2.5 protein (Figure 4B). Essentially all N1ICD/c-myg-tag-positivecells showed Nkx2.5 indicating that Notch1 signaling was a crucial modulator of Nkx2.5



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expression (Figure 4C; Online Figure VII). Thus, Notch1 activation was characterized byCSC commitment with the acquisition of the early immature myocyte phenotype; 65% ofthese cells retained their replicative potential and were positive for the cell cycle markerKi67 (Online Figure VII). Cycling cells positive for N1ICD and Nkx2.5 exhibited a thinlayer of sarcomeric proteins typical of transit amplifying cells (Figure 4C and 4D). At 10days, CSCs overexpressing N1ICD maintained a poorly developed cell phenotype: theyexpressed Nkx2.5, were cycling and could be passaged. Conversely, control cells increasedin size, rarely were Nkx2.5-positive and showed enhanced accumulation of contractileproteins (Figure 4E and 4F). Cell apoptosis was not affected by N1ICD overexpression(Online Figure VII).

Collectively, these observations suggest that the Notch1 receptor is a major regulator of theearly differentiation of neonatal CSCs and their transition to amplifying myocytes. Thequestion was then whether a similar mechanism of myocyte generation was operative invivo and contributed to postnatal myocardial growth.

Notch1 and Myocardial GrowthThe in vitro results support the notion that the expression of Notch1 receptor on the plasmamembrane of CSCs is linked to a permissive state in which the multipotentiality of CSCs ispreserved. Conversely, the translocation of N1ICD to the nucleus leads to the preferentialcommitment of CSCs to the myocyte lineage with the acquisition of the amplifying state.This control of myocyte growth offered the opportunity to determine whether CSCs and theNotch1 pathway are involved directly in the formation of myocytes after birth contributingto physiological cardiac hypertrophy.

The increase in myocardial mass after birth can occur by four mechanisms: a) Hypertrophyof the cardiomyocytes formed in prenatal life; b) Proliferation and hypertrophy ofcardiomyocytes present at birth; c) Generation of new cardiomyocytes by activation andcommitment of resident CSCs; and d) A combination of these cellular processes. Scenario aand b do not require the involvement of CSCs.

At birth, Notch1 was detected in CSCs while cardiomyocytes did not express theextracellular domain of Notch1 or other Notch isoforms (Online Figure VIII; see also Figure3C and 3D; Online Figure IV). In view of this differential distribution of Notch1 in CSCsand myocytes, stimuli activating or inhibiting Notch1 signaling impact exclusively onprimitive cells and do not affect preexisting myocytes. The presence of N1ICD in myocytenuclei was by necessity restricted to parenchymal cells derived from CSC differentiation.Thus, to determine whether Notch1 activation in CSCs was an important variable ofpostnatal myocardial growth with the formation of a large myocyte progeny, the Notchsystem was inhibited in newborn mice for a period of 5–7 days.

Notch receptor function was markedly attenuated by administration of a γ-secretase inhibitorwhich opposed the translocation of N1ICD to the nucleus and the initiation of transcriptionof Notch-dependent genes.5 The 5–7 day interval was chosen because this phase of cardiacdevelopment is characterized by a substantial increase in work load on the myocardium andmaximum increase in heart weight-to-body weight ratio. However, the γ-secretase inhibitormay affect Notch-independent pathways, imposing a cautious interpretation of the collectedresults. Unfortunately, inducible cardiomyocyte-specific N1ICD null mice would not clarifythe role of CSCs during physiological hypertrophy after birth because Notch1 deletionwould affect cells distal to CSCs. Similarly, the use of a c-kit promoter to target Notch1 inCSCs would involve c-kit-positive cells in multiple organs obscuring its effects on the heart.



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If CSC-mediated myocyte formation is required in postnatal myocardial growth, a potentialreduction in cardiomyogenesis may result in severe alterations in ventricular anatomy andfunction. In comparison with control animals injected with DMSO, mice exposed to the γ-secretase inhibitor diluted in DMSO showed echocardiographically a reduction in fractionalshortening and ejection fraction together with ventricular dilation and relative wall thinning(Figure 5A through 5C; Online Movies I and II). Five of the 37 mice injected with thevehicle died over a period of 7 days. Following treatment with the γ-secretase inhibitor, therate of animal mortality increased 3.3-fold, from 13% to 43%; 34 of the 79 treated mice diedduring this interval (Figure 5D). The high mortality of mice receiving DMSO was related toits unspecific toxic effects and was consistent with previous reports.11 Although we did notestablish the cause of death in these mice, echocardiographic measurements performed at 3,5 and 7 days did not show cardiac alterations characteristic of a lethal myopathy (OnlineFigure IX). In this regard, no further animal death was observed over a period of two weeksfollowing suspension of DMSO administration. Conversely, mice exposed to the γ-secretaseinhibitor continued to die after cessation of treatment (see below).

In all animals, echocardiographic data were complemented with morphometricmeasurements in tissue sections which allowed the computation of chamber volume andwall thickness at a uniform sarcomere length of 2.2 μm. This approach was employed due tothe inevitable limitations inherent in echocardiography of the neonatal mouse heart and theimpossibility to define with precision end-diastole and end-systole. The anatomical resultsstrengthened the in vivo observations, documenting a striking degree of ventricular dilationand thinning of the wall with γ-secretase inhibition (Figure 5E and 5F). Thus, alterations inNotch receptor function in newborns lead to a dilated myopathy which develops rapidly andresults in high animal mortality, emphasizing the importance of Notch signaling in thephysiological maturation of the heart.

Notch Inhibition and CardiomyogenesisTo assess the functional role of N1ICD in the generation of cardiomyocytes from activationand commitment of CSCs, the coexpression of c-kit, N1ICD and Nkx2.5 was determined.These markers together with the presence of sarcomeric proteins identify the pool ofmyocyte progenitors-precursors in the myocardium. Amplifying myocytes represent thesubsequent downstream step of the differentiation program; they are cycling c-kit-negativecells which contain transcription factors and cytoplasmic structures typical of maturing cells.3,5,12 Since myocytes do not possess the Notch1 extracellular domain, N1ICD in their nucleidemonstrates their origin from Notch1-positive CSCs.

During embryonic development from E10 to the end of gestation, ~55% of myocyte nucleiexpressed N1ICD and Nkx2.5 and this value decreased to ~30% at birth (Figure 6A through6D). The colocalization of N1ICD and Nkx2.5 in myocyte nuclei was confirmed by spectralanalysis (Online Figure X). The progressive decline in the fraction of N1ICD-positivemyocytes was paralleled by a comparable reduction in the number of cells expressingNkx2.5, which comprised ~30% of myocytes at birth (Figure 6E). This value diminishesfurther in the adult myocardium where the presence of Nkx2.5 is restricted to newly formedsmall myocytes derived from the differentiation of Notch1-positive CSCs.5 In newborns,myocytes negative for N1ICD were larger and contained greater amounts of sarcomericproteins (Figure 6F).

At completion of γ-secretase inhibition, 1 week after birth, the fraction of c-kit positiveN1ICD-positive cells, including CSCs and myocyte progenitors-precursors, decreased 45%(Figure 7A through 7C). The number of Nkx2.5 early committed CSCs was severelyaffected, resulting in a dramatic attenuation in the pool of cells potentially responsible forcardiomyogenesis. Ki67-positive and N1ICD-positive myocytes were reduced 62% and



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66%, respectively (Figure 7D through 7H). Hes1 and Nkx2.5 were also downregulated(Figure 7I). Thus, inactivation of the homeostatic control of cardiac growth by blockade ofthe proteolytic cleavage of Notch1 interfered with myocyte formation favoring the structuraland functional manifestations of a dilated myopathy.

Notch Inhibition and Physiological HypertrophyThe expansion in cardiac mass postnatally is accomplished by increases in wall thicknessand chamber diameter which reflect the adaptation of the myocardium to the increases inpressure and volume loads on the developing heart. The gross morphological indices ofventricular dimensions, wall thickness and chamber diameter, are the expression of acombination of parameters which include: a) Number of myocytes across the wall; b)Average myocyte cross sectional diameter and length; and c) The volume composition of themyocardium.

Interference with CSC activation by γ-secretase inhibition affected the formation of amyocyte progeny leading to a dilated myopathy with disproportionate increase in leftventricular chamber volume (see Figure 5). Attenuation in the generation of cardiomyocytesled to a reduction of heart weight. Decreased LV weight was dictated by a 54% smallernumber of cardiomyocytes which were 55% larger (Figure 8A). Cell apoptosis wascomparable in control and treated mice at 3 and 7 days (Online Figure XI), excluding thatcell death had a major impact on cardiac pathology and ventricular dysfunction. Importantly,the number of lineage negative CSCs decreased 30% in treated mice (Online Figure XI),indicating that the primary cause of the dilated cardiomyopathy was related to loss anddefective differentiation of resident stem cells.

The hypertrophic response of the remaining myocytes was unable to compensate for theeffect on myocyte formation. Myocyte hypertrophy was mostly mediated by celllengthening with little change in cell diameter. As a consequence, wall thinning wasproduced by a decrease in the mural number of cardiomyocytes while cell lengthening wasimplicated in the expansion of cavitary volume (Figure 8B). Conversely, the number ofcapillary profiles per mm2 of myocardium did not decrease as a result of myocytehypertrophy, suggesting that the coronary vasculature grew in proportion to the increase inmyocyte size (Figure 8B). Although ~10% ECs expressed the Notch1 receptor in control andtreated mice, γ-secretase inhibition did not affect their proliferation rate measured by Ki67labeling (Online Figure XI). Collectively, these data indicate that defects in myocyteformation in combination with disproportionate cell lengthening promote alterations inventricular anatomy favoring the transition from physiological to pathological hypertrophy,early in the maturation of the mouse heart.

Evolution of Postnatal Myocardial GrowthAn important question concerned the reversibility of the dilated myopathy and the recoveryof the functional and structural integrity of the myocardium. In rodents, myocyte formationand hypertrophy are dramatically enhanced during postnatal maturation up to the time ofweaning, decreasing considerably thereafter. To establish the consequences of Notchinhibition during the first week of life on the subsequent growth of the heart and organismwell-being, animal mortality was evaluated for an additional 2 weeks. Moreover, thecharacteristics of the heart in terms of cardiac anatomy, myocyte size and number and extentof cell replication were determined at this interval together with the analysis ofechocardiographic parameters.

The first week after cessation of γ-secretase inhibition, 11 of the 31 treated animals diedwhile only one of the remaining 20 mice did not survive the subsequent week. These data



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suggest that the effects of Notch blockade began to fade away ~7 days following the lastadministration of the drug. Conversely, only one of the 18 control mice injected with DMSOdied during the same interval (Figure 8C). These striking changes in the survival of treatedmice at 3 weeks were characterized by restoration of fractional shortening, ejection fractionand cardiac size measured echocardiographically (Figure 8D). Anatomically, wall thicknessand chamber volume returned to almost normal values. This structural recovery wasmediated by myocyte hypertrophy and a significant increase in myocyte number; cellgrowth reduced by 52% the difference in the total number of myocytes present at one weekbetween treated and untreated hearts (Figure 8E and 8F). The fraction of cycling myocytesand their mitotic index were 53% and 95% higher in treated than in control mice,respectively (Figure 8G).

DiscussionFindings in the current study indicate that the postnatal development of the heart is largelydependent on the generation of a myocyte progeny which is not present at birth but resultsfrom growth and differentiation of resident CSCs. Inhibition of this process by blockade ofNotch signaling affects the increase in myocyte number and cardiac mass necessary tosustain the work load of the growing organism and the changes in the patterns of blood flowand peripheral resistance dictated by the switch from the fetal to the adult circulatorysystem. Preexisting myocytes undergo cellular hypertrophy but this response cannot preventthe onset of a dilated myopathy and high mortality. These observations underscore the rolethat CSCs have in the physiological acquisition of the adult heart phenotype. The notion thatmyocyte hypertrophy alone is responsible for the maturation of the heart after birth1 has tobe reconsidered.

Plasticity of the Forming MyocardiumThe developing heart is a dynamic organ in which myocyte hypertrophy, myocyte death and,as shown here, de novo formation of myocytes from differentiation of CSCs all contribute tothe changes in cardiac size and shape that occur shortly after birth until myocardial loadinghas stabilized in the young adult organism. A significant fraction of myocytes expressesearly in life the senescence-associated protein p16INK4a and undergo apoptosis suggestingthat a subset of these cells has a rather short lifespan13 and myocyte renewal is an importantregulatory process of cardiac maturation. Although interference with cardiomyogenesis byinhibition of the Notch pathway has a powerful negative effect on the structure and functionof the growing heart, a similar mechanism is operative in adulthood and aging. Spontaneousmutations of the c-kit receptor with nearly 80–90% reduction in its tyrosine kinase activity14

are coupled with a marked attenuation in the adaptation of the heart to ischemic myocardialinjury.15 Similarly, the aging myopathy typically shows loss of CSCs in animals andhumans16,17 and, in animal models, the aging cardiac phenotype may be rescued by delayingCSC senescence or activating the residual pool of functionally competent CSCs.17,18

Results here in the neonatal heart by employing a loss of function strategy strengthen thenotion that the modulation of cardiac growth physiologically and pathologically is mediatedby the ability of cardiomyocytes to hypertrophy but, most importantly, by the inherentcapacity of CSCs to expand rapidly the myocyte pool. Activation and growth of CSCsincrease the muscle compartment to degrees that cannot be achieved by cellular hypertrophyalone.19,20 The CSC is the regulatory cell that controls physiological hypertrophy andconditions pathological hypertrophy and cardiac failure.

CSCs expressing the stem cell antigen c-kit have previously been found in the neonatalmouse heart.21,22 From birth to one month of age, the increase in myocyte volume and thegrowth of the coronary vasculature lead to an apparent decline in the number of CSCs in the



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adult organ.21,22 However, the relative proportion of CSCs is rather constant, one CSC/30,000 cells, in animals12 and humans.23 This value is comparable to the frequency ofhematopoietic stem cells (HSCs) in the bone marrow, one HSC/10,000–100,000 cells.24,25

Additionally, hematopoietic markers have been identified in c-kit-positive CSCs, suggestingthat HSCs migrate from the bone marrow to the heart contributing to the formation ofmyocytes and coronary vessels.22,26 Our results do not support this possibility; CSCs arenegative for CD34, CD45 and prominin, challenging the contribution of the bone marrow tothe development of the mouse heart. Recently, unrealistic values of c-kit-positive HSCs(9%) and CSCs (0.5%) have been reported26 questioning the validity of the approach andinterpretation of the data.

Notch1 and CSC FateOur in vitro and in vivo results indicate that the molecular pathway that controls the clonalformation of myocytes3 and the transition from undifferentiated CSCs to amplifyingmyocytes involves primarily the activation of Notch1. The ability of Notch to modulate thedevelopmental decision of progenitor cells is not unique to the heart. The Notch system isinvolved in cell fate specification by maintaining the stemness or favoring the commitmentof primitive cells.27 In the latter case, the function of Notch is restricted to one of the twosteps involved in stem cell differentiation: the early phase of acquisition of a specific cellphenotype or the late phase of cell maturation. In analogy with the heart, skeletal muscleregeneration involves initially a Notch-dependent response in which the activation ofsatellite cells gives rise to a population of highly replicating intermediate progenitors thatexpress myogenic proteins.42 The transition from the proliferative to the quiescent state ofmaturing myotubes does not occur until Notch signaling is downregulated and the Wntpathway sets in motion its downstream targets.

The relevance of Notch in the balance between cell proliferation and differentiation has beendocumented in the embryonic heart.29 Notch1 controls the evolution from the primitivemyocardial epithelium to the trabecular and compact myocardium. Prolonged Notch1activation leads to excessive cardiomyocyte proliferation with formation of ectopictrabecular structures.29 As shown in our study, this effect of Notch1 persists after birth andreplicating myocytes derive from activation of CSCs and upregulation of Nkx2.5. However,it cannot be excluded that N1ICD-negative cardiomyocytes constitute a population ofmyocytes formed independently from differentiation of CSCs. Importantly, the expressionof Notch1 in CSCs and the heterogeneity in the distribution of N1ICD and Nkx2.5 inreplicating myocytes may reflect temporally regulated modulation of Notch1 signaling. Thisprocess may involve the transition from undifferentiated Notch1-positive CSCs to earlycommitted N1ICD-positive myocytes and, then, to functionally competent N1ICD-negativematuring parenchymal cells.

Consistent with our observations, the proliferative state of immature cardiomyocytes in vivohas been shown to depend on sustained upregulation of the Notch pathway and theintracellular localization of the cytosolic and nuclear fragments of Notch1.30 Replication ofneonatal myocytes in vitro is enhanced by exposure to Jagged 1 and blocked by γ-secretaseinhibition, suggesting that these cells express Notch1. However, the presence of Notch1 onthe myocyte membrane appears to be an in vitro phenomenon, as detected by us (not shown)and others.30 N1ICD dictates the acquisition of the cardiomyocyte phenotype in EPCs andthis response is enhanced by co-culture with neonatal myocytes secreting Jagged1.31

Neonatal myocytes possess Notch1 transcripts but RT-PCR assay cannot distinguishbetween the extra and intracellular domain of the receptor, failing to prove that Notch1 isactually expressed on the sarcolemma.31 In lower organisms, Notch inhibits cardiomyocyteformation and differentiation while in mammals, Notch1 deletion results in myocardialhypoplasia.32 These findings may indicate that Notch controls cardiogenesis at multiple



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stages, from the initial phase of mesoderm commitment to the final step of myocytegeneration and maturation.32,33 Differences in Notch function are likely dependent on theinvolvement of different receptor isoforms and ligands, the cell in question, the context andtiming of the process.

Notch1, Nkx2.5 and CardiomyogenesisDuring embryonic development, the homeodomain factor Nkx2.5 occupies an upstreamposition in the hierarchy of the cardiac regulatory genes, driving directly or indirectly theexpression of multiple genes involved in heart formation.34 Homozygous Nkx2.5 nullembryos are characterized by arrest of cardiac growth and death between 9.5 and 11.5 daysof prenatal life35 pointing to the essential role of Nkx2.5 in cardiac morphogenesis.However, chimeric embryos with variable contribution of Nkx2.5-null myocytes show thatthe severity of the cardiac abnormality is proportional to the fraction of Nkx2.5-null cellspresent in the developing heart.34 The normal phenotype of chimeric hearts including 15%Nkx2.5-null cells suggests that the concomitant expression of Nkx2.5 in all myocytes is notrequired for the proper development of the heart. These findings are consistent with theprogressive decrease in the number of Nkx2.5-positive myocytes from the embryonic to theadult heart shown here and previously.35 The heterogeneous distribution of Nkx2.5 is,however, in conflict with the notion that this transcription factor is indispensable for themaintenance of the differentiated state of cardiomyocytes. To reconcile these conflictingobservations, the possibility has been raised that Nkx2.5 may promote the synthesis andsecretion of paracrine factors that support the survival and the maturation of the surroundingembryonic myocytes.34 A similar phenomenon may be operative in the neonatal and adultmyocardium. Activation of the Notch1 receptor may transactivate directly the Nkx2.5 genein a subset of CSCs promoting indirectly the commitment of adjacent primitive cells andpreserving the terminally differentiated phenotype of neighboring adult cardiomyocytes.This mechanism may contribute to the control of cardiac homeostasis when old dyingcardiomyocytes are replaced by new cells which co-express Nkx2.5 and the active fragmentof Notch1.5

Mutations of genes of the Notch system cause cardiac abnormalities in geneticallyengineered mice32 and humans.36 Notch signaling promotes the epithelial-to-mesenchymaltransition that is crucial for the formation of valvular structures37 and alterations of Notchsignaling in progenitor cells of the second heart field result in abnormalities of the aorticarch and outflow tract.38 Since inhibition of Notch1 leads to the development of a dilatedmyopathy in neonatal mice, the hypothesis may be advanced that Notch1 is involved in theetiology of idiopathic dilated cardiomyopathy (IDC) in children. Dilated cardiomyopathy isthe most common cause for cardiac transplantation in children but its etiology is unknown in~65% of cases.39 Our findings raise the possibility that pediatric IDC may involve defects ofNotch1 in CSCs with severe attenuation in the generation of cardiomyocytes.

In conclusion, the Notch1 receptor and its ligand Jagged1 are critical determinants of thecommitment of neonatal CSCs to the myocyte lineage, the generation of amplifyingmyocytes and the formation of a differentiated progeny. Shortly after birth,cardiomyogenesis counteracts ongoing myocyte death and expands the pool of parenchymalcells in the growing organ, fundamental factors in the progressive maturation of the heartand the acquisition of the adult cardiac phenotype. When this cascade of events is alteredphysiological cardiac hypertrophy is prevented, maladaptive processes ensue and a lifethreatening dilated myopathy supervenes.

Novelty and Significance

What Is Known?



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• Notch1 is involved in stem cell fate specification in multiple organs.

• Cardiac stem cells (CSCs) in the adult heart express the Notch1 receptor andactivation of the Notch pathway plays a critical role in the acquisition of themyocyte lineage.

• Postnatal cardiac growth is mediated mostly by hypertrophy of preexistingmyocytes.

What New Information Does This Article Contribute?

• The neonatal heart contains a population of c-kit-positive CSCs that express theNotch1 receptor.

• The increase in cardiac mass postnatally involves largely the generation ofcardiomyocytes mediated by activation of the Notch1 receptor in CSCs.

• Inhibition of the Notch1 pathway in CSCs early after birth impairscardiomyogenesis leading to the development of a dilated myopathy innewborns.

Our work challenges the traditional view that postnatal cardiac growth is induced only byhypertrophy of preexisting fetal cardiomyocytes in the absence of new myocyteformation. In the current study we have documented that the physiological maturation ofthe heart is a complex process in which cardiomyogenesis plays a critical role in theacquisition of the adult heart phenotype. A myocyte progeny, not present at birth, isderived from growth and differentiation of resident c-kit-positive cardiac stem cells(CSCs) stored in niches. Commitment of CSCs to the myocyte lineage with thegeneration of transit amplifying myocytes is controlled by the activation of the Notch1receptor pathway. Blockade of Notch signaling affects the increase in myocyte numberand cardiac mass resulting in a dilated myopathy with high mortality. Restoration ofmyocyte regeneration rescues the failing heart and animal survival. These observationsunderscore the important function that CSCs have in the adaptation of the heart to theabrupt changes in myocardial loading which occur during the transition from the fetal tothe adult circulatory system in mammals.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsSources of Funding

This work was supported by NIH grants.

Abbreviations and Acronyms

CSCs Cardiac stem cells

N1ICD Notch1 intracellular domain

Ecs Endothelial cells

SMCs Smooth muscle cells

α-SA α-sarcomeric actin

α-SMA α-smooth muscle actin



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vWf von Willebrand factor

HSCs Hematopoietic stem cells

IDC Idiopathic dilated cardiomyopathy

EPCs endothelial progenitor cells

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Figure 1.CSCs in the neonatal heart are clustered in niches and form a progeny of transit amplifyingmyocytes. A through D: Atrial (A and B) and ventricular (C and D) niches at 7 days afterbirth composed of clusters of undifferentiated c-kit-positive CSCs (green). Some of thesecells express Nkx2.5 alone (white; arrows) or Nkx2.5 together with a thin layer of α-SA(red; arrowheads). E and F: Small dividing myocytes show phospho-H3 (green) in theirnuclei. Metaphase chromosomes are illustrated at higher magnification in the insets.



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Figure 2.Myocytes undergo cytokinesis in the neonatal heart. A through D: Myocyte cytokinesis; thecolocalization of phospho-H3 (A and C; green) and Aurora B (B and D; white) is apparent.E: Dividing myocytes express phospho-H3 (white) and are c-kit-negative; an adjacent CSCis c-kit-positive (green; arrow). Myocytes are labeled by α-SA (red).



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Figure 3.Activation of Notch1 in CSCs by Jagged1 leads to translocation of N1ICD to the nucleusand upregulation of Nkx2.5. A and B: CSCs form functionally competent myocytes. Sino-atrial nodal-like CSC-derived myocyte with spontaneous action potentials. Depolarizingsteps to −40 mV and +10 mV reveal the lack of the fast Na+ current (INa) and the presenceof Ca2+ current (ICa), respectively (A). Action potentials in a ventricular-like CSC-derivedmyocyte stimulated at 1 and 2 Hz. Depolarizing steps activate both INa and ICa (B). C andD: LV myocardium at 6 days after birth in which the CSCs included in the squares areshown at higher magnification in the adjacent panels. CSCs are positive for c-kit (green) andNotch1 (red). E and F: c-kit-positive CSCs (arrows) are in contact with myocytes andJagged1 is distributed at the interface between cardiomyocytes and CSCs (insets,arrowheads). N1ICD (yellow dots) is present in CSC nuclei.



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Figure 4.N1ICD overexpression in CSCs promotes the formation of a pool of replicating myocytes.A: Quantitative RT-PCR for target genes of Notch and cardiovascular genes. Expression isshown as change with respect to CSCs infected with EGFP-lentivirus only. B: Fraction ofNkx2.5-positive CSCs in the absence (control) and presence of N1ICD overexpression(N1ICD). Results are mean±SD. *P<0.05. C: CSCs overexpressing N1ICD (green) arepositive for Nkx2.5 (red) and possess a thin layer of α-SA (white). D: Ki67 labeling (yellow)of mitotic (arrows) and non-mitotic nuclei of amplifying myocytes (α-SA, red) derived fromdifferentiation of N1ICD-CSCs. E and F: Myocytes with modest (E) and significant (F)accumulation of contractile proteins (α-SA, red; Nkx2.5, green).



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Figure 5.Notch inhibition induces a dilated myopathy. A and B: B-mode and M-modeechocardiography of vehicle (left) and γ-secretase inhibitor (right) injected mice. C and D:γ-secretase inhibition led to ventricular dilation, depressed fractional shortening, ejectionfraction and increased mortality. E: With respect to a control heart (left panel), ventriculardilation and wall thinning are apparent in γ-secretase inhibitor treated mice (central and rightpanels). F: Effects of γ-secretase inhibition on cardiac anatomy. Results are mean±SD.*P<0.05.



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Figure 6.The fraction of embryonic and neonatal cardiomyocytes co-expressing N1ICD and Nkx2.5decreases with maturation. A through C: In myocyte nuclei, N1ICD (white) is coexpressedwith Nkx2.5 (yellow) at E10 (A), E11.5 (B) and E19 (C). D: Fraction of myocytes positivefor N1ICD. Results are mean±SD. *P<0.05 vs E10, E11.5 and E19. E: Fraction of Nkx2.5-positive myocytes after birth. F: At 7 days after birth, N1ICD-negative myocytes are largerthan N1ICD-positive cells. Laminin (green) defines the cell boundary.



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Figure 7.Notch inhibition interferes with cardiomyogenesis in vivo. A and B: CSCs positive (A) andnegative (B) for N1ICD (white). C: γ-secretase inhibition decreased the number of CSCsexpressing N1ICD. D through H: Notch blockade attenuated the number of myocyteslabeled by Ki67 or N1ICD qualitatively (D–G) and quantitatively (H). Results are mean±SD. *P<0.05. I: Quantitative RT-PCR for Hes1 and Nkx2.5. Expression is shown aschanges with respect to vehicle injected mice.



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Figure 8.Reversibility of the effects of Notch inhibition on myocytes, cardiac anatomy and function.A and B: Effects of γ-secretase inhibition at one week on heart weight, myocyte number,volume, cross-sectional area (CSA) and length. Number of myocytes across the LV wall andcapillary density are also shown. C: Mortality rate following cessation of γ-secretaseinhibition. D through F: M-mode echocardiogram at 21 days, i.e., 2 weeks after the lastadministration of the γ-secretase inhibitor, together with fractional shortening, ejectionfraction and chamber diameter (D). Cardiac anatomy, myocyte number and volume (E andF) and the fraction of cycling and dividing myocytes (G) are also shown.



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Inhibition of Notch1-Dependent Cardiomyogenesis Leads to a Dilated Myopathy in the Neonatal Heart