968 NATURE MEDICINE • VOLUME 6 • NUMBER 9 • SEPTEMBER 2000

NEWS & VIEWS

Listening for hoof beats in heart beatsDisruption of the Sp1-related transcription factor HF-1b leads to alterations in the cardiac conduction system that resultin ventricular arrhythmias and sudden death. What does this tell us about how ventricular cardiomyocytes differentiate

into the specialized cells that form the heart’s conductance system?

JEFFREY ROBBINS1 & GERALD W DORN II2

THE ROLES OF hypertension and coronaryartery disease in heart failure are well

established, but it is only within the last 10years that we have begun to understandthe specific molecular mechanisms thatunderlie cardiomyocyte-based heart dis-ease. A seminal paper in 1990 first estab-lished that naturally occurring mutationsof the cardiac contractile protein β-myosinheavy chain caused familial hypertrophiccardiomyopathy1. Other disease-causingmutations in genes encoding the contrac-tile apparatus have since been identified,currently totaling 150 mutations in 9 dif-ferent cardiac sarcomeric proteins(http://www.angis.org.au/Databases/Heart/).Mutations in ion channels, which regulateelectrical conductance of the cardiomy-ocyte, have been previously shown tocause diseases such as arrhythmogenicsudden cardiac death (SCD). For instancemutations in a sodium channel encodedby HERG (ref. 2) or in potassium channelgenes3 can produce the long QT syn-drome.

Mutations in genes encoding transcrip-tion factors lead to an interesting subset ofcongenital heart malformations, such asthe atrial-septal defects caused by muta-tions in the homeobox transcription factor gene NKX2.5 (ref. 4), and the cardiac malformations characteristic ofHolt–Oram syndrome that result from mu-tations in the T-box transcription factorTBX5 (ref. 5). In the 31 August issue ofCell, Nguyen-Tran et al.6 report that disrup-

tion of the gene encoding an SP1-relatedtranscription factor in mice leads to deathfrom SCD.

To maintain a rhythmic beat through-out the entire cardiac mass, the heart con-tains a specialized system for precisespatial and temporal transmission of theelectric impulse (Fig. 1). During a normalheartbeat, the atria contract about 1/6 of asecond before the ventricles, allowing forventricular filling. Subsequently, almostall portions of the ventricles contract si-multaneously, allowing efficient ejection.Cardiac contraction is dependent upon aspecialized excitatory and conductive sys-tem. This includes the sinus node, or sino-atrial (SA) node where the initial impulseis generated, the internodal pathways,which conduct the impulse from the SAnode to the atrioventricular (AV) node,and the AV node itself, where the impulseis delayed for approximately 90 ms whilethe atria empty. The conductive systemalso includes the Purkinje fibers, whichhave both left and right bundles, such thatthe impulse is conducted to all areas of theventricles almost simultaneously (Fig. 1).

Nguyen-Tran et al. observed that micedeficient for the transcription factor HF-1bexhibited normal cardiac structure andfunction, but often succumbed to SCD

(ref. 6). As sudden death is often caused byarrhythmogenic events, the authorslooked to the conduction system to ex-plain the apparent phenotype. Their find-ings shed light on the molecular eventsinvolved in the development of thesehighly specialized electrical conductingcells from cardiomyocyte progenitors, anddescribe a novel mechanism for primarypro-arrhythmic events in the heart.

This study is unique because of thespecificity of the cardiac defects detectedin the HF-1b null mice. Nguyen-Tran et al.reported that although heterozygous mu-tants are normal, the surviving homozy-gotes develop ventricular arrhythmias,increased action potential duration and other electrical abnormalities6.Determining this mechanism of death re-quired a formidable and unprecedentedtechnical tour de force in longitudinalmurine physiology. This included pro-grammed electrical stimulation and con-tinuous ambulatory 24 hour EKGmonitoring—procedures that are standardtechniques for evaluating arrhythmic po-tential in human patients, but are chal-lenging to perform in mice.

The authors used whole cell patchclamp techniques to detect pro-arrhyth-mogenic events such as alterations in theduration and dispersal of the action poten-tial6. They discovered that the density ofthe Ikslow rectifier current, a current that isat least partially responsible for the overallregulation of the action potential, was de-

antigen recognized by cytotoxic T lymphocytes.Immunity 10, 673–679 (1999).

11. Minev, B. et al. Cytotoxic T cell immunity againsttelomerase reverse transcriptase in humans. Proc.Natl. Acad. Sci. USA 97, 4796–4801 (2000).

12. Bendandi, M. et al. Complete molecular remissioninduced by patient-specific vaccination plus gran-ulocyte-monocyte colony-stimulating factoragainst lymphoma. Nature Med. 5, 1171–1177(1999).

Department of Experimental Transplantation & ImmunologyMedicine Branch,Division of Clinical SciencesNational Cancer InstituteBethesda, Maryland 20892 USAEmail: kwak@mail.ncifcrf.gov

prominent role in these cancer thera-peutics.

1. Kugler, A. et al. Regression of human metastaticrenal cell carcinoma after vaccination with tumorcell-dendritic cell hybrids. Nature Med. 6,332–336 (2000).

2. Nair, S.K. et al. Induction of cytotoxic responsesand tumor immunity against unrelated tumorsusing telomerase reverse transcriptase RNA trans-fected dendritic cells. Nature Med. 6, 1011–1017(2000).

3. Brocker, T. et al. Targeted expression of major his-tocompatibility complex class II moleculesdemonstrates that dendritic cells can induce neg-ative but not positive selection of thymocytes invivo. J. Exp. Med. 185, 541–550 (1997).

4. Ludewig, B. et al. Immunotherapy with dendriticcells directed against tumor antigens shared withnormal host cells results in severe autoimmune

disease. J. Exp. Med. 191, 795–803 (2000).5. Siegal, F.P. et al. The nature of the principal type 1

interferon-producing cells in human blood.Science 284, 1835–1837 (1999).

6. Rissoan, M.C. et al. Reciprocal control of T helpercell and dendritic cell differentiation. Science 283,1183–11186 (1999).

7. Biragyn, A., Tani, K., Grimm, M.C., Weeks, S. &Kwak, L.W. Genetic fusion of chemokines to a selftumor antigen induces protective, T-cell depen-dent antitumor immunity. Nature Biotech. 17,253–258 (1999).

8. Kim, N.W. et al. Specific association of humantelomerase activity with immortal cells and can-cer. Science 266, 2011–2013 (1994).

9. Weng, N.P. et al. Regulated expression of telom-erase activity in human T lymphocyte develop-ment and activity. J. Exp. Med. 183, 2471–2479(1996).

10. Vonderheide, R.H. et al. The telomerase cataliticsubunit is a widely expressed tumor-associated

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NATURE MEDICINE • VOLUME 6 • NUMBER 9 • SEPTEMBER 2000 969

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creased. Immediately after depolarization(the initial upstroke of the action poten-tial), the potassium channel responsiblefor this current begins to lose its ability toconduct current outward, causing an earlyfall in membrane conductance. The selec-tive reduction of this repolarizing currentcould explain both the increase inthe action potential’s duration andthe alterations observed in its dis-persal pattern.

The cells that carry current inthe conduction system can beidentified by their distinct physiol-ogy, histology and protein expres-sion pattern. Nguyen-Tran et al.examined expression of the con-duction system-specific proteinmarkers connexin 40 and minK,and demonstrated that in HF-1bnull mice, connexin 40 expressionwas decreased and aberrantly local-ized, whereas minK expression wasupregulated6. The authors con-cluded that HF-1b is required fordevelopment of a specialized sub-population of ventricular car-diomyocytes, and that deficits inthe conduction system of nullmice were due to defects in the spe-cialized myocytes that make upthe Purkinje fibers.

A previous analysis of HF-1b nullmice by Supp et al. reported earlyinexplicable mortality, with only a30% survival rate by 7 days afterbirth7. Nguyen-Tran et al. report asurvival rate of 41%, and revealthat the mice die from arrhythmicevents6. The slight differences inmortality rates between the twostudies may be due to differencesin gene targeting constructs. Theconstruct used by Supp et al. dis-rupted a region of HF-1b that en-coded three zinc-binding domains,and the authors reported that theywere able to detect partial tran-scripts, raising the possibility ofhypo-allelism. In contrast, the con-struct used by Nguyen-Tran et al. disruptedthe gene at the start codon6, presumablypreventing any production of HF-1b tran-script or protein (although this was notrigorously documented). Complete dis-ruption increases the potential for oppor-tunistic compensation by relatedtranscription factors of the SP1 family.Another explanation for differences in sur-vival rate between the two studies is thatthe partial transcript of Supp et al. acts in adominant negative fashion, leading to in-

creased mortality in these mice.Although the data are intriguing, there

is no evidence that HF-1b is directly in-volved in the conduction cell lineage.Over-expression of HF-1b in ventricularcardiomyocytes did not result in a de-tectable phenotype6, implying that other

factors, which may or may not interactwith HF-1b, are involved. Lineages areoften determined by both extrinsic andintrinsic factors and this is probably truefor the conduction system. Analysis ofmice with ventricular myocyte-specificdisruptions in HF-1b should revealwhether the defects observed are due to aspecific requirement for this factor in con-duction cell differentiation or the sys-temic effect of deleting a multifunctionaltranscription factor.

The conduction system develops frommyocytes adjacent to the developing en-domyocardium and coronary arteries.Studies have shown that over-expressionof both preproendothelin-1, the precursorfor endothelin-1 (ET-1), and its convertingenzyme, ECE-1, is sufficient to induce car-

diomyocytes to express Purkinjefiber markers ectopically8. ET-1 isproduced by endothelial cells inthe heart and can act in a paracrinefashion to induce Purkinje fiber de-velopment. Although ET-1 recep-tors are expressed throughout thedeveloping myocardium, ECE-1 isnot and its expression may deter-mine the location of ET-1 signalingin the heart. Whether HF-1b is up-stream or downstream of theseevents remains to be determined.

Like most studies, the findingsof Nguyen-Tran et al. raise manyquestions and will prompt new in-vestigative efforts. Do HF-1b muta-tions exist in the humanpopulation and are they associatedwith cardiac conductance defects?What other SP1 family membersare expressed by conduction sys-tem cells and are their expressionpatterns altered in the HF-1b nullmice? How is the lineage-inducingpathway mechanistically orga-nized? How important are theparacrine versus autocrine arms ofthe pathway? Do the promoter re-gions of the minK and connexin 40genes contain regulatory elementsthat bind HF-1b, or do its actionsdepend upon co-factors?

Just as hundreds of mutationshave been associated with humanheart disease, there are hundredsof genetically manipulated mousemodels of cardiac diseases.However, a mouse is not a man,and genetic alterations that areknown to cause lethal ventriculararrhythmias in human patientshave not had similar effects in

mice9. Similarly, there are mouse modelsof cardiac hypertrophy and dilated car-diomyopathy that have no parallel tohuman disease10. There is no doubt that bymaking in vivo gain or loss of functionstudies possible, genetic manipulation inthe mouse has proven to be a powerful ap-proach for achieving fundamental mecha-nistic insights into pathophysiologicprocesses and, at times, genetic manipula-tion has also mimicked aspects of humandisease. However, every student of medi-

Fig. 1 Cardiac conduction system. The sinus node (sometimescalled the sinus-atrial node) serves as the heart’s pacemaker,emitting an impulse that results in an action potential. The cellsin the node have almost no contractile elements but are con-nected directly to the atrial fibers, so that the action potentialspreads immediately into the atrial cardiomyocytes and is trans-mitted through the entire atrial muscle mass. However, propaga-tion occurs more rapidly through three specialized bundles ofatrial muscle called the internodal pathways, which contain, inaddition to the atrial cardiomyocytes, specialized conductivecells. This has the net effect of transporting the conductive im-pulse to the atrial-ventricular (A-V) node within 30 msec. There isa delay of ∼ 130 msec in the A-V node and bundle system duringwhich time the atria can contract, filling the ventricles. The con-ducting impulse is then propagated through the specializedPurkinje cells of the conduction system. These are large cells thatare able to transmit the action potential at 2–4 m/s, a rate that issix times that of the normal ventricular cardiomyocyte. The im-pulse is transmitted through the entire Purkinje fiber systemwithin 30 msec, ensuring that a rhythmic and concerted ejectionof blood from the ventricles take place as the endocardial cellsand finally the epicardial cells are stimulated.

Sinusnode

Internodalpathway

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Inferiorvena cava

Superiorvena cava AV

node

AVbundle

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Leftatrium

Rightatrium

Rightventricle

Leftbundlebranch

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cine learns that, in considering a diagno-sis, “when you hear hoof beats, look forhorses since zebras are rare.” Likewise, increating exotic, zebra-like genetic models,which have some of the characteristics ofhuman disease, let us not forget the com-mon horse.

1. Geisterfer-Lowrance, A.A. et al. A molecular basis forfamilial hypertrophic cardiomyopathy: a beta cardiacmyosin heavy chain gene missense mutation. Cell 62,999–1006 (1990).

2. Wang, Z. et al. Functional effects of mutations inKvLQT1 that cause long QT syndrome. J. Cardiovasc.Electrophysiol. 10, 817–826 (1999).

3. Ackerman, M.J. The long QT syndrome: ion channeldiseases of the heart. Mayo Clin. Proc. 73, 250–269(1998).

4. Schott, J.J. et al. Congenital heart disease caused by

mutations in the transcription factor NKX2-5. Science281, 108–111 (1998).

5. Basson, C.T. et al. Mutations in human TBX5 causelimb and cardiac malformation in Holt-Oram syn-drome. Nature Genet. 15, 30–35 (1997).

6. Nguyen-Tran, V.T.B. et al. A novel genetic pathwayfor sudden cardiac death via defects in the transitionbetween ventricular and conduction system cell lin-eages. Cell, (in the press).

7. Supp, D.M., Witte, D.P., Branford, W.W., Smith, E.P.& Potter, S.S. Sp4, a member of the Sp1-family of zincfinger transcription factors, is required for normalmurine growth, viability, and male fertility. Dev. Biol.176, 284–299 (1996).

8. Takebayashi-Suzuki, K., Yanagisawa, M., Gourdie,R.G., Kanzawa, N. & Mikawa, T. In vivo induction ofcardiac Purkinje fiber differentiation by co-expressionof preproendothelin-1 and endothelin converting en-zyme-1. Development 127, 3523–3532 (2000).

9. Kupershmidt, S. et al. Replacement by homologousrecombination of the minK gene with lacZ reveals re-

striction of minK expression to the mouse cardiacconduction system. Circ. Res. 84, 146–152 (1999).

10. Sussman, M.A. et al. Myofibril degeneration causedby tropomodulin overexpression leads to dilated car-diomyopathy in juvenile mice. J. Clin. Invest. 101,51–61 (1998).

1Division of Molecular Cardiovascular BiologyChildren’s Hospital Research Foundation, 3333 Burnet AvenueCincinnati, Ohio 45229-3039Email: jeff.robbins@chmcc.org2Division of CardiologyUniversity of Cincinnati231 Bethesda AvenueCincinnati, Ohio, 45267-0542Email: dorngw@ucmail.uc.edu

970 NATURE MEDICINE • VOLUME 6 • NUMBER 9 • SEPTEMBER 2000

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How many factors are required to remodel bone?Overexpression of two members of the AP-1 transcription factor family has dramatic effects on bone development,raising the question: why are there so many molecules that affect bone remodeling (pages 980–984 and 985–990)

GERARD KARSENTYBONE REMODELING IS the physiologicprocess used by vertebrates to main-

tain a constant bone mass and to renewbone throughout life. It is comprised oftwo phases, resorption of pre-existingbone tissues by the osteoclasts, followedby de novo bone formation by the os-teoblasts. Thus, the regulation of bone re-modeling involves molecules thatcontrol differentiation and functionof two very different and complex celltypes. In this issue, Jochum, et al.1 andSabatakos, et al.2 respectively reportthat over-expression of Fra1 or ∆FosB,two members of the AP1 sub-familyof leucine zipper-containing tran-scription factors, result in osteosclero-sis, or unregulated bone formation byosteoblasts. These two elegant andcomplementary studies raise a num-ber of questions regarding the tran-scriptional control of osteoblastdifferentiation, a process that is notcompletely understood.

Bone formation and/or bone re-sorption is known to be affected by al-terations in expression levels of AP1transcription factor family members.For example, transgenic mice overex-pressing c-fos develop osteosarcomas,and osteoblast tumor, whereas c-fosdeficient mice develop osteopetrosisdue to an early arrest in osteoclast differen-tiation3. Interestingly, this latter pheno-type can be rescued by over expression ofFra1, a Fos-related protein that lacks anidentifiable transactivation domain4.Analysis of Fra1-deficient mice indicates

that Fra-1 is not required for osteoblast orosteoclast differentiation in vivo1, yet Fra1over-expressing mice have increased os-teoblast differentiation resulting in an os-

teosclerotic phenotype.The same is true in the case of mice that

over-express ∆FosB, a naturally occurringtruncated form of FosB that arises from al-ternative splicing of the fosB transcript.The phenotypic abnormalities of these

two different transgenic mouse models arevery similar; the increase in osteoblast dif-ferentiation and bone formation in bothmodels is cell autonomous and does notresult in an increase in osteoclast differen-tiation or in bone resorption in vivo.

In view of this spectacular phenotype,the lack of an identifiable transactivation

domain in Fra1 is puzzling. Again thesame is true for the ∆FosB over-ex-pressing mice, as ∆FosB proteins lackthe carboxy-terminal domain thatconstitutes the major transactivationdomain of FosB (ref. 5). Two generalmechanisms could be envisioned toexplain the phenotypic abnormali-ties observed in these two mousemodels, both involving the ability ofFra1 and ∆FosB to heterodimerizewith several leucine zipper-contain-ing proteins (Fig. 1). The first modelpredicts that over expression of ei-ther of these proteins in osteoblastsincreases their chances of het-erodimerization with another nu-clear protein required for osteoblastdifferentiation. This new partnerneed not to be the physiological part-ner of Fra1 or ∆FosB in wild type os-teoblasts, but the resultingheterodimer may have a higher affin-ity for DNA or an enhanced ability to

activate transcription (Fig. 1a). The mostlikely partner of Fra1 and ∆FosB would bea leucine zipper-type of transcription fac-tor critical for osteoblast differentiation.In any case, this putative protein partneris not Cbfa1, because Fra1 transgenic mice

Fig. 1 Two models can account for the effect of ∆FosBand Fra1 over-expression. a, Over-expression of either∆FosB or Fra1 (orange circle) favors osteoblast differentia-tion because these factors heterodimerize with a transcrip-tional activator (green circle) of osteoblast differentiation.This heterodimer could have a higher affinity for DNAand/or a higher transcriptional activity. b, alternativelyFra1 and ∆FosB over-expression could titer out, throughheterodimerization, a transcriptional inhibitor (blue circle)of osteoblast differentiation.

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