re-expression of proteins involved in cytokinesis during cardiac hypertrophy

E X P E R I M E N T A L C E L L R E S E A R C H 3 1 3 ( 2 0 0 7 ) 1 2 7 0 – 1 2 8 3

ava i l ab l e a t www.sc i enced i rec t . com

www.e l sev i e r. com/ loca te /yexc r

Research Article

Re-expression of proteins involved in cytokinesis duringcardiac hypertrophy

Preeti Ahujaa,1, Evelyne Perriarda, Thierry Pedrazzinib, Shinji Satohc,Jean-Claude Perriarda, Elisabeth Ehlerd,e,⁎aInstitute of Cell Biology, ETH Zürich-Hönggerberg, CH-8093 Zürich, SwitzerlandbDepartment of Medicine, University of Lausanne Medical School, CH-1011 Lausanne, SwitzerlandcDivision of Cardiology, Kyushu Medical Centre, National Hospital Organization, 1-8-1 Jigyohama, Chuo-ku, Fukuoda 810-8563, JapandThe Randall Division of Cell and Molecular Biophysics, King's College London, Room 3.28A, New Hunt's House, Guy's Campus,London SE1 1UL, UKeThe Cardiovascular Division, King's College London, London SE1 1UL, UK

A R T I C L E I N F O R M A T I O N

⁎ Corresponding author. The Randall DivisionGuy's Campus, London SE1 1UL, UK. Fax: +44

E-mail address: [email protected] Current address: Department of Medicine

0014-4827/$ – see front matter © 2007 Elsevidoi:10.1016/j.yexcr.2007.01.009

A B S T R A C T

Article Chronology:Received 26 October 2006Revised version received15 January 2007Accepted 16 January 2007Available online 27 January 2007

Cardiomyocytes stop dividing after birth and postnatal heart growth is only achieved byincrease in cell volume. In some species, cardiomyocytes undergo an additional incompletemitosis in the first postnatal week, where karyokinesis takes place in the absence ofcytokinesis, leading to binucleation. Proteins that regulate the formation of the actomyosinring are known to be important for cytokinesis. Here we demonstrate for the first time thatsmall GTPases like RhoA along with their downstream effectors like ROCK I, ROCK II andCitron Kinase show a developmental stage specific expression in heart, with high levelsbeing expressed in cardiomyocytes only at stages when cytokinesis still occurs (i.e.embryonic and perinatal). This suggests that downregulation of many regulatory andcytoskeletal components involved in the formation of the actomyosin ring may beresponsible for the uncoupling of cytokinesis from karyokinesis in rodent cardiomyocytesafter birth. Interestingly, when the myocardium tries to adapt to the increased workloadduring pathological hypertrophy a re-expression of proteins involved in DNA synthesis andcytokinesis can be detected. Nevertheless, the adult cardiomyocytes do not appear to dividedespite this upregulation of the cytokinetic machinery. The inability to undergo completedivision could be due to the presence of stable, highly ordered and functional sarcomeres inthe adult myocardium or could be because of the inefficiency of degradation pathways,which facilitate the division of differentiated embryonic cardiomyocytes by disintegratingmyofibrils.

© 2007 Elsevier Inc. All rights reserved.

Keywords:CardiomyocytesCytokinesisCytoskeleton

of Cell and Molecular Biophysics, King's College London, Room 3.28A, New Hunt's House,20 7848 6435.

(E. Ehler).and Physiology, UCLA School of Medicine, Los Angeles, CA 90095-1760, USA.

er Inc. All rights reserved.

mailto:[email protected]

http://dx.doi.org/10.1016/j.yexcr.2007.01.009

1271E X P E R I M E N T A L C E L L R E S E A R C H 3 1 3 ( 2 0 0 7 ) 1 2 7 0 – 1 2 8 3

Introduction

During embryogenesis heart mass mainly increases by celldivision of cardiomyocytes, a process that is called hyperplasia[1] with only some cellular growth [2]. Embryonic cardiomyo-cytes are able to divide in their differentiated state [3], unlikeskeletal muscle cells, where proliferation and differentiationare mutually exclusive processes. After birth cell divisionceases in the mammalian heart and growth is achievedexclusively by increase in cell size, i.e. hypertrophy [4]. Thistransition from hyperplasia to hypertrophy after birth ismarked by binucleation in some species, where cardiomyo-cytes completely loose their ability to finish cytokinesis [5]. Inrodents for example, the accumulation of binucleated cardio-myocytes starts around day four and by the third postnatalweek 85–90% of the cardiomyocytes have two nuclei [5]. Atpresent the cause for this uncoupling of karyokinesis fromcytokinesis is not known.

In contrast, cardiomyocytes from lower vertebrates canstill divide postnatally [6]. In adult newt and zebrafish, hearttissue can even be regenerated after injury [7]. However, inmammals the general consensus is that cardiomyocytes inadults are postmitotic and do not retain any proliferationpotential; for a review, see [8–10]. No myocardial regenerationseems to occur in diseases or injuries that lead to cardiomyo-cyte loss. Besides, primary myocardial tumors are rarelyobserved in adults [11]. Recently, the existence of residentcardiac stem cells was reported [12–14], but it seems thatdespite these cells displaying progenitor properties, theircontribution to cardiac repair in damaged hearts is minimalunder normal situations (for a review, see [15]). The inability ofadult mammalian cardiomyocytes to divide accounts for amajor difficulty in restoration of function to the damagedheart [16]. Induction of DNA synthesis as a response to stressor other factors has been described, [17,18], yet cell division isno longer observed, resulting in multiple nuclei or polyploidy[11]. So far nothing is known about the expression of proteinsthat regulate cytokinesis in the diseased heart.

Cytokinesis is the final step of cell division. It is responsiblefor equal partitioning and separation of cytoplasm betweendaughter cells to complete mitosis [19]. There are four majorevents contributing to cytokinesis which include (i) determi-nation of the division site, (ii) actomyosin based contractilering formation followed by ingression, (iii) midbody formationand (iv) cell separation (for a review, see [20]). A number ofpossible key players such as small Rho GTPases and theireffectors ROCK I and ROCK II, LIMK, Citron kinase and septinsare important for regulating the formation of the actomyosinring [21].

Rho GTPases are known to play an important role in a widespectrum of cell activities, including cell division (cytokinesisin particular), cell migration and cardiomyocyte hypertrophy[22–24]. Of all these RhoA ismost crucial for cytokinesis [25]. Innumerous cell types, inactivation of RhoA leads to profounddefects in cytokinesis, and in most cases cleavage furrowformation is completely blocked [26]. Rho-kinase/ROK/ROCK,one of the downstream targets for RhoA GTPase, for a reviewsee [27], is known to lead to an increase in myosin light chainphosphorylation, which activates themotor activity ofmyosinand leads to the contraction of the actomyosin ring [28].

LIM kinase, another substrate of ROCK, contributes indir-ectly to assembly of the contractile ring by stabilizingfilamentous actin due to phosphorylation of cofilin/ADF [21].Therefore, expression of phosphorylated cofilin can also betaken as an indication for cytokinetic activity.

To find out whether the observed uncoupling of karyokin-esis and cytokinesis in cardiomyocytes after birth can becorrelated to changes in expression levels of proteins thatare known to be important during cytokinesis, we checkedfor the expression levels of actomyosin ring regulating pro-teins during cardiac development. We therefore investigatedthe expression of Rho GTPases and their downstreameffectors like ROCK I, ROCK II and p-cofilin and found thatwhile high levels can be detected in embryonic cardiomyo-cytes, a dramatic downregulation of expression is observedafter birth, which might account for the uncoupling ofkaryokinesis from cytokinesis at this stage. Since severalproteins that are only expressed in the embryonic heartshow upregulation in the hypertrophic heart [8], we nextchecked for re-expression of cytokinesis regulating proteinsin several animal models for hypertrophic cardiomyopathy.Our analyses revealed that there is re-expression of proteinsthat are involved in DNA synthesis and cytokinesis in myo-cardium in response to hypertrophy, despite the fact thatmitosis is usually not resumed under these conditions. Wesuggest that upregulation of the expression of cytokinesisregulating proteins is together with increased DNA synthesisone way of cardiomyocytes to respond to pathological stress,but that they are prevented from going through mitosis,possibly by their elaborate cytoskeleton in form of themyofibrils.

Materials and methods

SDS–PAGE and immunoblotting

SDS samples from whole ventricles of mice of the appropriatestageswerepreparedasdescribed before [29]. TheSDS sampleswere run on 7.5%, 15% or 22% polyacrylamide minigelsdepending on the molecular weight of the proteins (Bio-Rad,Glattbrugg, Switzerland) and immunoblotting was performedas described previously [29].

Isolation and culture of embryonic and neonatal ratcardiomyocytes (ERC and NRC)

Primary cultures from embryonic (day 14) and neonatal ratventricles were prepared and maintained as described pre-viously [3]. Hypertrophy was induced by treatment with Phen-ylephrine (Sigma) at a concentration of 100 μM, withIsoproterenol (Sigma) at 10 μM and Norepinephrine (Sigma)in serum-free medium at 10 μM for 72 h just before fixation orbefore preparing SDS samples. The calpain inhibitor NCO-700(Alexis) was used at 20 μM for 3 h just before fixation.

Immunofluorescence on cryosections and cells

Cryosections were prepared as described previously [2].The fixation, ensuing immunofluorescence procedures, and

Fig. 1 – Expression levels of cytokinesis-associated proteinsduring heart development. Cell lysates from embryonic heart(E14, lane 1), day of birth (P0, lane 2), day eight (P8, lane 3),adult mouse ventricular muscle (lane 4) and adult mousekidney tissue lysate (positive control, lane 5) were analyzedby immunoblotting for expression of several proteinsinvolved in control of the actomyosin ring: RhoA, Rac1,Cdc42, p-Cofilin, ROCK II, ROCK I and PCNA as a karyokineticprotein. Equal amounts of heart tissue were loaded asdemonstrated by usingα-cardiac actin as loading control. Allmarkers associated with karyokinesis and cytokinesis areexpressed at high levels in samples from embryonic heart,start to get downregulated after birth and are in most casescompletely absent in the adult heart. Equal amounts of totalproteins were loaded in each lane, but the intensity of thebands obtained for different antibodies does not reflect therelative amounts of these proteins compared to each other.

1272 E X P E R I M E N T A L C E L L R E S E A R C H 3 1 3 ( 2 0 0 7 ) 1 2 7 0 – 1 2 8 3

analysis by confocal microscopy were carried out as describedpreviously [3].

Antibodies and fluorescent reagents

The monoclonal mouse (mM) antibodies against RhoA, Cdc42and Proliferating Cell Nuclear Antigen (PCNA) were obtainedfrom Santa Cruz Biotechnology (Santa Cruz, CA). The mMantibodies against Rac1 and ROCK II were from BD Bios-ciences. The polyclonal rabbit (pR) antibody against phos-phorylated-Cofilin, the pR antibody against ROCK I, the mManti-M-protein antibody (clone AA259, IgA), the pR anti MyBP-C and the pR anti α-skeletal actin were generously donatedby James Bamburg (Colorado State University, Colorado),Didier Job (INSERM, France), Dieter O. Fürst (Bonn University,Germany), Mathias Gautel (King's College London, UK) andChristine Chaponnier (University of Geneva, Switzerland),respectively. The monoclonal rat anti α-tubulin (cloneYOL1/34) was from Abcam, UK, the pR antibodies against phos-phorylated histone H3 and against Polo like kinase were fromUpstate Biotechnology. The mM anti α-cardiac actin wasobtained from Progen (Heidelberg, Germany), the mM anticalpain-1 from Calbiochem (Juro, Luzern, Switzerland). ThemM anti non-muscle myosin IIB was from Chemicon (Teme-cula, California, USA). The pR antibodies against atrialnatriuretic factor (ANF) were purchased from ANAWA (Wan-gen, Switzerland). The pR anti Cullin-3 was characterized inthe lab of Matthias Peter. The secondary antibody andfluorescent reagent combinations for the triple immunofluor-escence stainings and for immunoblotting were as describedpreviously [29].

Densitometric and statistical analysis

X-ray films were scanned in gray scale at 300 dpi with anHPscanjet Iicx hardware (Hewlett Packard Company, Palo Alto,USA) and submitted to densitometric analysis using Image Jsoftware (National Institute of Health, Washington, USA). Thedensitometric values represent the mean of the gray intensityof all the pixels present in a selected area. For normalization, adensitometric analysis of a selected reference was performed,e.g. cardiac actin content. Blots were performed in triplicateand statistical analysis was done using Excel software (Micro-soft, Redmond, USA). Data are given as mean±standarddeviation. Bars in the graphs represent standard errors anddifferences analyzed with a two-tailed T test with a P valuebelow 0.05 being considered significant.

Binucleation

Nuclei were stained with 4,6-diamidino-2 phenylindole-2 HCl(DAPI). The percentage of mononucleated and binucleatedcells was counted from a sample of 200 isolated myocytesfrom 2 individual sets using confocal microscopy images.

Hypertrophic samples

Hypertension induced one-kidney, one clip (1K1C) mouseheart and sham-operated mouse heart [30], isoproterenoltreated and its control mouse heart, angiotensin II transgenic

mouse heart alone and treated with isoproterenol [31] wereprovided by Dr. Pedrazzini.

Dahl salt resistant rats (DR), sensitive (DS) and DS ratstreated with Y-27632 heart extracts [32] were provided by Dr.Satoh. All animals were maintained according to the localrules.

Results

Expression and localization of several actomyosinring-associated proteins during heart development

To find out whether neonatal rodent cardiomyocytes stopdividing due to the lack of their entire cytokinesis machineryor by downregulation of an individual regulatory component,the expression pattern of several Rho GTPases and theirdownstream effectors at different stages of mouse heartdevelopment was investigated by immunoblotting withspecific antibodies (Fig. 1). We analyzed RhoA, Rac1, Cdc42,p-Cofilin, ROCK II, ROCK I as well as the DNA synthesismarker PCNA in samples from embryonic day 14, day of birthP0, P8 and adult mouse heart along with adult mouse kidneytissue lysate as a positive control. In the embryonic heart (Fig.1, lane 1), high levels of expression of all these proteins could


be found, which get downregulated by the first postnatalweek and were mostly absent in adult heart (Fig. 1, lane 4).This suggests that the Rho GTPases and their downstreameffectors tested here are only highly expressed in cardiomyo-cytes that are still able to divide and do not seem to play amajor role in the adult heart.

Since members of the Rho family are known to beinvolved in regulating the actin cytoskeleton in general, we

Fig. 2 – Localization of three different actomyosin ring-associatedduring division. Confocal micrographs of ERC stained for the smagreen in overlay), for the myofibrillar protein myosin binding protubulin to assess the stage of mitosis (panels J, K, L and blue in oundergoing cytokinesis. All three small GTPases show strong micytokinesis, as indicated by arrows in panels (G, H and I). Scale b

investigated their subcellular distribution in cardiomyocytesby triple immunofluorescence. Primary cultures of embryonicrat cardiomyocytes were chosen for analysis by confocalmicroscopy since they still possess a proliferative potentialwhile being fully differentiated at the same time [3]. Cardio-myocytes undergoing cytokinesis were identified by stainingwith an antibody specific for tubulin (Figs. 2J, K, L; blue inoverlay), which finally concentrates in the midzone region

proteins in cultured embryonic rat (E14) cardiomyocytes (ERC)ll GTPases RhoA, Rac1, Cdc42 (panels G, H, I, respectively, andtein-C (MyBP-C) (panels D, E, F and red in overlay) and forverlay). Double arrows in panels A, B and C point to the cellsdbody localization in embryonic cardiomyocytes undergoingar represents 10 μm.

Fig. 3 – Re-expression of cytokinesis-associated proteins inhypertrophy. Immunoblot with different antibodies againstdifferent cytokinesis regulating proteins on SDS samples ofisoproterenol-treated and 1K1C mouse heart together withtheir respective controls. All the cytokinetic proteins showincreased expression: RhoA, Cdc42, Rac1, ROCK II and ROCKI, including PCNA in hypertrophic samples of isoproterenoltreated for 1 week (lane 2) and 2 weeks (lane 3) compared toits control (lane 1) and in the 1K1C heart (lane 5) in contrast toits control (lane 4), sham-operated heart. Equal amounts ofheart tissuewere loaded as demonstrated by usingα-cardiacactin expression as loading control.


before the cells pinch off to form daughter cells (indicated bydouble arrows in the Figs. 2A, B, C). Myosin binding protein C(MyBP-C), which is a component of the A-band, served as amyofibrillar marker (Figs. 2D, E, F; red in overlay). MyBP-C islocalized in a cross-striated pattern in interphase, prophaseand metaphase cardiomyocytes; however, the localization ofMyBP-C becomes completely diffuse during anaphase andthe cross-striated localization pattern begins to re-establishonly after cytokinesis [3]. We found that RhoA, Cdc42 andRac1 were only expressed at high levels during latetelophase of cardiomyocytes and showed a locationrestricted to the midbody region (Figs. 2G, H, I, indicated byarrow).

This suggests that similar to observations in most mam-malian cells [33], the small GTPases RhoA, Cdc42 and Rac1 arealso involved during cytokinesis in cardiomyocytes. Thus,their downregulation may be responsible for the uncouplingof cytokinesis from karyokinesis in cardiomyocytes afterbirth.

Re-expression of cytokinesis-associated proteins in heartduring hypertrophy

After having shown the downregulation of proteins involvedin DNA synthesis and cytokinesis after birth and theirabsence in adult heart, we now wanted to explore the pos-sibility of cardiomyocytes making an attempt to initiate celldivision activity again during heart disease. It is believed thatthere is a partial progress through the cell cycle underhypertrophic conditions in an attempt of the myocardium tocope with stress [17,18,34]. However, it is not known whetherany of the known regulators of cytokinesis is re-expressedduring hypertrophy. A failure to upregulate these proteinsmight provide a simple explanation why adult cardiomyo-cytes do not divide despite the DNA synthesis activity duringdisease. To test, whether cytokinetic markers also reappearin heart during the hypertrophic response, well-establishedin vitro and in vivo mouse and rat models for hypertrophywere investigated.

Two different mouse models for cardiac hypertrophy wereanalyzed initially for expression of cytokinesis regulatingproteins, isoproterenol stimulated mice and one-kidney, oneclip (1K1C) mice [30,35,36]. Both animal models are wellcharacterized and represent with increased heart weight tobody weight ratios as listed in Supplementary Table 1. Wholeheart samples were analyzed by immunoblotting withantibodies against RhoA, Cdc42, Rac1, ROCK I, ROCK II andPCNA. A marked increase in the expression levels of all theseproteins could be observed in the different hypertrophymodels compared to control animals (Fig. 3; compare lanes 2and 3 with lane 1, and lane 5 with lane 4, respectively). Theseresults indicate that lack of cardiomyocyte division in adultmammalian disease cannot be explained by a generalabsence of the cytokinesis regulating machinery. This re-expression may be a specific response to stress or maysimply mirror the re-expression of known marker proteinsfor hypertrophy that are usually only present in the fetalventricle and also upregulated during disease such as ANF, α-skeletal actin and β-myosin heavy chain (for a review, see[8]).

Localization of upregulated cytokinetic protein markers indiseased heart

To verify that the observed upregulation of expression levelsof proteins that are involved in the regulation of cytokinesisoccurs in the cardiomyocytes themselves and is not simplydue to a fibrotic reaction or increased infiltration of inflam-matory cells, we next performed immunofluorescence analy-sis of triple-stained cryosections from hypertrophy models.Only the results for the 1K1C mice are depicted, but similarobservations were also made in the other mouse models forhypertrophy that we studied (Fig. 4, data not shown).Cryosections of wild-type (Figs. 4A–D, I–L) and 1K1C mousehearts (Figs. 4E–H, M–P) were triple stained with antibodies toM-protein (Figs. 4B, F, J, N; red in overlay) to identify thecardiomyocytes, RhoA (Figs. 4C, G; green in overlay A, E) andCdc42 (Figs. 4K, O; green in overlay I, M), as cytokinesis-associated proteins, while expression of non-muscle myosinheavy chain IIB (Figs. 4D, H, L, P; blue in overlay) was used tovisualize cardiac fibroblasts and other non-muscle cells [37].Single confocal sections revealed a complete absence ofexpression of RhoA and Cdc42 in wild-type sections (Figs. 4C,K) in cardiomyocytes as well as in non-muscle myosin heavychain IIB positive non-muscle cells. In sections from 1K1Chearts (Figs. 4G, O), a drastic increase in the signal for RhoA aswell as Cdc42was detected (Figs. 4G, O; green signal in overlay)compared to sham-operated animals. This can in part beattributed to a fibrotic reaction as indicated by the increased

Fig. 4 – Confocal micrographs illustrating the localization of upregulated cytokinetic proteins in the diseased heart.Cryosections of sham-operated (panelsA–Dand I–L) and 1K1Cmouse hearts (panels E–HandM–P) triple stainedwith antibodiestoM protein (panels B, F, J, N and red in overlay) to visualize cross striations in the cardiomyocytes, RhoA (panels C, G and greenin overlay A, E) and Cdc42, respectively (panels K, O and green in overlay I, M) as cytokinesis-associated protein marker andnon-muscle myosin heavy chain IIB (panels D, H, L, P and blue in overlay) to visualize cardiac fibroblasts. Single confocalsections reveal that sections from control hearts (panels C and K) do not show expression of cytokinetic proteins, while 1K1Csections (panels G and O) display a drastic increase in signal for both RhoA (panel G) and Cdc42 (panel O) in cardiomyocytes(arrows in G, O) along with increased fibrosis as demonstrated by the overlay with the non-muscle myosin IIB signal (panels Hand P). Scale bar represents 10 μm.


amount of non-muscle cells stained with the non-musclemyosin heavy chain IIB antibody (Figs. 4H, P; blue signal inoverlay). However, there is clearly also a signal for RhoA andCdc42 in the cytoplasm of the cardiomyocytes in theseconfocal micrographs, which is partially associated with themyofibrils (arrows in 4G and O). We were unable to detectcardiomyocytes that are positive for DNA synthesis markerssuch as phosphorylated histone H3; however, also no positivefibroblasts could be identified in these sections, indicatingthat the time period of expression of thesemarkersmay be tooshort to retrieve positive cells in tissue culture sections (datanot shown, see also [11]). Our results show that while theupregulated expression of cytokinesis-associated proteinssuch as RhoA and Cdc42 at the level of the immunoblot ispartially due to the contribution of non-muscle cells, asignificant amount of the increase is due to the cardiomyo-cytes themselves.

Angiotensin II overexpressing mice as hypertrophic model

To determine whether increased expression levels of cytokin-esis-associated proteins can also be observed in a genetic

mouse model for hypertrophic cardiomyopathy, we usedtransgenic mice that overexpress angiotensin II in a cardio-myocyte specific fashion (Ang II mice; [31]). These micedevelop hypertension-independent cardiac hypertrophywith-out signs of fibrosis [31]. Ventricular extracts from Ang II micewere probed by immunoblot with antibodies against cytokin-esis-associated proteins. As expected, there was a markedincrease in the expression of all these proteins in cell lysates ofAng II mouse hearts (Fig. 5A), when compared with theirrespective controls (Fig. 5A). Analysis of the expression levelsof bona fide hypertrophymarkers revealed that the increase inexpression in cytokinesis-associated proteins is well corre-lated with increased levels of ANF and α-skeletal actin (Fig.5A). All these results imply that the upregulation of expressionof cytokinesis-associated proteins can indeed be considered anovel marker for hypertrophy and is not just due to fibrosis.

Hypertension-induced hypertrophy of Dahl salt sensitive ratswith and without Y-27632 treatment

Experiments on cardiomyocytes in culture have shown thatinterference with the ROCK signaling pathway downstream of

Fig. 5 – Upregulation of cytokinesis-associated proteins inangiotensin II overexpressing mice and Dahl Salt sensitiverats. (A) Immunoblots on SDS samples of Ang II hearts wereprobed with antibodies against cytokinesis-associatedproteins; RhoA, Rac1, Cdc42, ROCK II and ROCK I. There is amarked increase in the expression levels of all the cytokineticproteins in cell lysates of Ang II hearts compared to controls,respectively. Equal amounts of heart tissue were loaded asshown by using α-cardiac actin as a loading control.(B) Immunoblot on heart samples from Dahl salt sensitive(DS) and resistant rats (DR) fed on high salt diet withantibodies against several cytokinesis related proteinmarkers. SDS samples of left ventricular tissue of (1) DR ratsas a normotensive control, (2) DS rats with no treatment, (3)DS rats treated with Y-27632, a selective ROCK inhibitor wereprobedwith RhoA, Cdc42, Rac1, ROCK II and ROCK I aswell asPCNA as a karyokinetic marker. DS rats without treatmentshowed high expression of all the cytokinetic markersincluding PCNA in comparison to DR rats, which served asnormotensive controls. DS rats treated with Y-27632 showeda slightly blunted response in their expression of cytokinesismarkers in contrast to DS rats with no treatment. Equalamounts of heart tissue were loaded; α-cardiac actin wasused as loading control.


Rho by using the relatively specific inhibitor Y-27632 can bluntthe hypertrophic response [38]. Recently, this beneficial effectwas also demonstrated in an in vivo model, the Dahl salt-sensitive (DS) rat [32]. This animal model develops hyperten-sion, thus promotingmyocardial hypertrophy, LV dilation andheart failure when fed on a high-salt diet [39]. Treatment of DSanimals on a high salt diet with Y-27632 leads to a significantlyreduced hypertrophic response and also improved contractilefunction; however, the degree of interstitial fibrosis wassimilar to untreated DS animals [32]. We reasoned thatventricular tissue from this animal model would be an idealway to test our hypothesis since (1) the interferencewith ROCKsignaling would allow us to investigate the effect on theexpression levels of other cytokinesis regulating proteins and(2) the similar degree of fibrosis between treated and untreatedanimals would be a good additional control to exclude changesin expression levels solely based on increased numbers offibroblasts. Immunoblot analysis was performed on leftventricular tissue lysates of (1) Dahl salt resistant (DR) rats asa normotensive control (Fig. 5B), (2) Dahl salt sensitive (DS) rats(Fig. 5B) and (3) DS rats treated with Y-27632 (Fig. 5B) on a highsalt diet using antibodies against different cytokinesis-asso-ciated proteins.

DS rats without treatment (Fig. 5B) that had developedhypertrophy displayed high expression of all the cytokineticmarkers in contrast to control DR rats (Fig. 5B, group 1). In Y-27632-treated DS rats, however, the expression of all cytokin-esis regulating proteins is significantly reduced compared toDS rats with no treatment, with the exception of Cdc42 (Fig. 5B,group 3). The differences in expression levels of cytokineticproteins in the Dahl salt induced hypertrophy model arequantified by densitometry analysis in Supplementary Fig. 1.

Therefore, reduced hypertrophy in Y-27632-treated DS ratsis accompanied by a reduced expression of cytokinesis-associated proteins despite the previous observation that theextent of fibrosis is comparable between treated anduntreated animals. This provides further confirmation thatthe upregulation is specifically correlated to hypertrophy.Interestingly, the expression of Cdc42 is unchanged in Y-27632-treated rats, suggesting that it belongs to an alternativepathway.

Re-expression of cytokinesis coupled proteins in an in-vitromodel of hypertrophy

Our results on in vivo models of hypertrophy suggest thatupon an insult adult rodent cardiomyocytes not only start tosynthesize DNA [18,40,41] but also upregulate the expressionof proteins that are involved in cytokinesis. Therefore,theoretically they should be all set for division, which never-theless does not appear to happen. We decided to explore thisquestion a bit more closely in a well-documented in vitromodel of hypertrophy using cultured cardiomyocytes [42],which are easier amenable to experimental interference.Primary cultures of neonatal rat cardiomyocytes (NRC) canbe induced to display a hypertrophic response when grown inserum-freemediumcontaining agents such as phenylephrine,isoproterenol or norepinephrine. Triplicate samples of therespective condition were probed on immunoblots withantibodies to cytokinesis-associated proteins such as RhoA,

Fig. 6 – Upregulation of cytokinesis-associated proteins in anin-vitro model of hypertrophy. Immunoblot on cell lysates ofprimary cultures of neonatal rat cardiomyocytes (NRC) grownin serum-free medium containing hypertrophy inducingagents; phenylephrine (100 μM), isoproterenol (10 μM) andnorepinephrine (10μM) or vehicle for 72 h. Triplicate sampleswere probed with antibodies against cytokinesis-associatedproteins; RhoA, Rac1, ROCK II, p-cofilin, polo like kinase andPCNA, p-His H3 as karyokinetic markers. A marked increasein the expression of all karyokinetic and cytokinetic proteinsin hypertrophic cardiomyocytes in contrast to controlneonatal rat cardiomyocytes was observed.


Rac1, ROCK II, p-cofilin, polo-like kinase (PLK) and for kar-yokinetic markers such as PCNA and phosphorylated histoneH3 (p-His H3). All the NRC cultures, where hypertrophy hadbeen induced, showed a marked increase in the expression ofRhoA, Rac1, ROCK II, p-cofilin, p-His H3 and PLK compared tocontrols (Fig. 6). The expression of the DNA synthesis markerPCNA, which was already quite high in unstimulated NRC,remained the same upon hypertrophic stimulation. Thequantification of the expression levels is shown in Supple-mentary Fig. 1. These results suggest that stimulation withhypertrophy inducing agents leads also in NRC to increasedexpression of proteins that are involved in the regulation ofcytokinesis. The consistently high levels of PCNA probablyreflect the fact that at this stage of development NRC gothrough another round of karyokinesis, leading to binucleatedcells.

While the induction of hypertrophy is well documented[42], the cellular effects have not been studied in great detail.Therefore, we stained stimulated NRC cultures with anti-bodies against RhoA to investigate its subcellular localization(Fig. 7). In addition, myofibril organization was visualized byphalloidin staining of F-actin and the nuclei with DAPI. Asreported previously, unstimulated NRC show a smaller cellsize. They express hardly any RhoA and mostly have onlyone nucleus. In NRC where hypertrophic growth wasinduced, a dramatic increase in the signal intensity forRhoA was seen in all three cases. Interestingly, the signal forRhoA was distributed completely diffuse throughout thecytoplasm and was not correlated with actin containingstructures such as the myofibrils. This is in contrast to theobservations in embryonic dividing cardiomyocytes, whereRhoA is specifically concentrated at the cleavage furrow(compare with Fig. 2A). Also in NRC that presumably just

underwent nuclear division (arrows in Figs. 7N, O, P), nosubcellular concentration of the RhoA signal can be detected,suggesting that cytokinesis is already deregulated in cardi-omyocytes at this developmental stage. Another remarkableobservation was the high incidence of binucleated cellsfollowing hypertrophic treatment (for quantification, see Fig.7Q). Thus, the signal in rodent heart to undergo karyokinesisin the first postnatal week can be mimicked also in vitro andseems solely due to a hypertrophic stimulus, which in theanimal is caused by increased workload after birth. Never-theless, complete divisions could not be seen in the cultures,again suggesting that upregulation of cytokinetic andkaryokinetic markers alone is not sufficient to push cardi-omyocytes through mitosis.

Expression of calpain-1 during heart development and effect ofcalpain inhibition on myofibril disassembly in embryonic ratcardiomyocytes

At present it is unclear, what regulates the disassembly ofmyofibrils that seems to be essential for cardiomyocytes toundergo cell division [3]. During catabolism in skeletalmuscle the myofibrils first have to be digested by calpains,followed by proteasomal degradation [43]. Initial experi-ments on cultured embryonic cardiomyocytes highlightedthe importance of proteasome activity for myofibril disas-sembly [3], but nothing is known about the role of calpain.Therefore, we initially investigated the expression levels ofcalpain-1 during heart development. Again we observed adevelopmental stage specific regulation, with prominentbands only being present in samples from the embryonicand neonatal heart and a downregulation of signal in theearly postnatal and adult heart (Fig. 8A). The endogenouscalpain inhibitor calpastatin shows no developmental stagespecific regulation of expression in heart (data not shown).Only a slight upregulation of expression levels of calpaincompared to wild-type littermates could be observed in theAng II mouse (Fig. 8B). In order to investigate any effect ofcalpain inhibition on myofibril disassembly, we again usedembryonic cardiomyocyte cultures and treated them withNCO-700, an inhibitor of calpain activity ([44]; Fig. 8C). Asreported previously, the signal for the Z-disk protein α-actinin is completely diffuse in control cultures at themetaphase stage, with some residual myofibrillar striationsstill being present in case of the M-band protein myomesin,indicating myofibril disassembly (Fig. 8C, control panels; [3]).In dividing embryonic cardiomyocytes that were treatedwith NCO-700, clear cross-striations can still be observed forα-actinin and myomesin, despite the fact that the cells havereached themetaphase stage, as indicated by the arrangementof the mitotic spindle, the condensed chromosomes and thepresence of phosphorylated histone H3. No cardiomyocytescould be observed that had progressed beyond metaphasestage. These experiments suggest that myofibril disassemblyis impaired in the absence of calpain activity and thereforecardiomyocytes are arrested in the metaphase state. The in-sufficient upregulation of calpain expression during hypertro-phy may be one explanation why adult cardiomyocytes fail todivide despite the upregulated expression of cell cycle andcytokinesis markers.


Neddylated cullin-3 is only expressed in the developing heartbut cannot be detected in hypertrophic samples

In an attempt to further characterize the role of ubiquitin-dependent proteolysis during heart development and disease,

we next investigated the expression of cullin-3, an ubiquitinE3 ligase. E3 ligases are responsible for bringing in substratespecificity into the ubiquitin–proteasome degradation system[45] and cullin family members are known to promote proteinubiquitination that is critical for cell cycle progression [46]. A

Fig. 8 – Calpain-1 expression during development and disease and role in myofibril disassembly. (A) Immunoblot withantibodies against calpain-1 on SDS samples from E14 (lane 1), P0 (lane 2); P8 (lane 3) and adult (lane 4) mouse ventricles showsa downregulation of calpain-1 expression after birth. Similar amounts of muscle tissue were loaded as demonstrated withα-cardiac actin as a loading control. (B) Immunoblot with antibodies against calpain-1 on SDS samples from control mouseventricles (7043) and ventricles from Ang II mice (8305, 8306, 181102) shows that compared to hypertrophic markers there isonly a slight upregulation of calpain-1 expression in the hypertrophic hearts. The blot had to be exposed for longer than thedevelopmental immunoblot to reveal the signal. (C) Embryonic rat cardiomyocytes were treated with the calpain inhibitorNCO-700 and the effect on myofibril disassembly was studied. Dividing cardiomyocytes were identified by being positive forphosphorylated histone H3 (green signal in overlay) and the organization of tubulin to a spindle (blue signal in overlay). Themyofibrils were visualized with antibodies against the M-band protein myomesin (A, B; red in top row) or the Z-disk proteinsarcomericα-actinin (C, D; red in bottom row). In control cells inmetaphase, theα-actinin signal is completely diffuse and alsothe myomesin signal is much less distinct than in neighboring interphase cells. NCO-700 treatment leads to an arrest ofmyofibril disassembly and also to an arrest of cell division in treated cardiomyocytes.


specific protein modification, called neddylation, regulatesthe activity of cullins positively [47]. Interestingly, analysis ofcullin-3 expression levels during heart development showedthat the neddylated highmolecular weight form is exclusivelydetected in the embryonic heart (Fig. 9A). While there is aslight upregulation of expression in hypertrophic samplesfrom Ang II mice, no neddylated cullin-3 can be seen (Fig. 9B).

Fig. 7 – Expression of RhoA is upregulated in primary cultures owith hypertrophy inducing agents. (A) Confocal micrographs of ngreen in overlay), for F-actin (panels E–H and red in overlay) andoverlay). Single confocal sections reveal that while control cardiosignal for RhoA (panel J–L) distributed throughout the cytoplasminducing agents for 72 h. Arrows in panels (N–P) point to binucleawith four nuclei. Scale bar represents 10 μm. The quantification opresented in panel Q.

These data suggest that it is actually a deregulation of theactivation of ubiquitin-dependent proteolysis, which mayprevent myofibril disassembly and ultimately results in aninability of adult cardiomyocytes to finish mitosis.

In conclusion, our results suggest that several proteins thatare involved in cytokinesis display a developmental stagespecific expression pattern in cardiomyocytes and are

f neonatal rat cardiomyocytes (NRC) following the treatmenteonatal rat cardiomyocytes stained for RhoA (panels I–L andfor DAPI to observe binucleation (panels M–P and blue inmyocytes display no signal (panel I); a drastic increase in thecan be seen following the treatment with the hypertrophyted cells and the asterisk in panel N points to a cardiomyocytef binucleation in hypertrophic neonatal rat cardiomyocytes is

Fig. 9 – Expression of the ubiquitin ligase cullin-3 duringheart development and disease. (A) Immunoblots ofventricular SDS samples of E14 (lane 1), P0 (lane 2), P8 (lane 3)and adult (lane 4) mice were probed with antibodies againstcullin-3. A double band, representing neddylated cullin-3 aswell as active cullin-3, can only be revealed in embryonicheart; in addition, a downregulation of cullin-3 levels duringdevelopment was observed. (B) Immunoblot on SDS samplesfrom ventricles of control (7043) and Ang II (8305, 8306,181102) reveal that cullin-3 expression levels do not changesignificantly in hypertrophic hearts and that also noactivation of cullin-3 seems to occur, as indicated by theabsence of neddylated cullin-3.


normally only expressed at high levels in the embryo, whenheart growth is still achieved by hyperplasia. Hypertrophicgrowth is associated with a re-expression of these proteins,which is nevertheless insufficient to drive cardiomyocytesthroughdivision, either in animalmodels for hypertrophy or instimulated cardiomyocytes in culture. It is proposed thatcalpain and cullin-dependent protein degradation pathways,which seem only to be active in the embryonic heart, fail torespond properly during hypertrophy and thus prevent theregulatedmyofibril disassembly and ensuing completemitosisin adult cardiomyocytes.

Discussion

One of the hallmarks of cardiac hypertrophy is the re-ex-pression of genes in the ventricle that are usually onlyexpressed during fetal heart development such as ANF, α-skeletal actin andβ-myosinheavy chain [8]. Also growthby celldivision, hyperplasia, is only observed in the fetal heart andour results presented here show that the expression ofproteins that are involved in cytokinesis, specifically in theregulation of the formation of the actomyosin ring, show adevelopmental stage specific expression in the heart and a re-expression during hypertrophy. The contractile ring wasinvestigated previously in postnatal cardiomyocytes from rat[48]. However, these localization studieswere restrictedonly toF-actin andnon-musclemyosin andnone of the other proteinsinvolved in cytokinesis was analyzed. A more recent studyinvestigated the localization of anillin, a known regulator ofthe cleavage furrow in dividing versus binucleating culturedcardiomyocytes and suggested that the failure of the latter do

undergo abscission is due to a defective focussing of anillin inthe mid-body region [49]. We demonstrate here for the firsttime that cardiomyocytes in situ show a developmental stagespecific expression also of other proteins that are coupledwiththe formation of the actomyosin ring such as RhoA, Cdc42,Rac1, ROCK-I, ROCK-II andp-cofilin,withhigh levels being onlyexpressed at stages when cytokinesis still occurs (i.e. embryo-nic and perinatal). Also kinases that are involved in theregulation of cytokinesis such as Polo-like kinase display thisdevelopmental expressionprofile [50] andwehavedeterminedpreviously that another family of small GTPases that arepresumably involved in the assembly of the actomyosin ring,the septins, are expressed at high levels only before birth [29].Thus, cardiomyocytes shut down their mitotic activity in thefirst week after birth, as documented by the downregulation ofcell cycle regulatory molecules [51,52] as well as of cytokinesisregulating proteins.

Expression of small actin regulating GTPases during heartdevelopment and disease

Analysis of the expression levels of the small actin regulatingGTPases RhoA, Rac1 and Cdc42 during heart developmentrevealed that they are only expressed at high levels in theembryonic heart and completely absent from healthy cardi-omyocytes in situ as demonstrated by immunofluorescenceanalysis. In non-muscle cells members of this family havebeen implicated to play several roles in regulating actomyosindynamics [22] but it appears that they are dispensable for theregulation of the actin cytoskeleton in mature cardiomyo-cytes. As far as their direct involvement in the regulation ofcytokinesis is concerned, it has been suggested that thedifferent small GTPases may have a cell-type specific, butpartially also redundant role inmammals (reviewed in [53]). Inthe heart, it has been shown previously that RhoA and Rac1can be directly involved in hypertrophic signaling processessince inhibition of RhoA signaling by drugs or expression ofdominant negative or constitutively active variants can eitherprevent or stimulate cell size increase in cultured cardiomyo-cytes [54]. We show here an upregulation of RhoA and Rac1expression in samples from hypertrophic hearts. This mayindicate that small GTPases initially get upregulated in theirexpression only as part of the switch back to a fetal geneexpression program, which also includes cytokinesis-asso-ciated proteins and may then aggravate the hypertrophicresponse by stimulation of additional downstream signalingpathways that are not related at all to the actin cytoskeleton[54]. The completely diffuse localization pattern that weobserved for RhoA following hypertrophic stimulation innon-dividing NRC, which is in marked contrast to its locationat the actomyosin ring in dividing embryonic cardiomyocytes,supports the interpretation that its involvement in theprogression of hypertrophy is completely separate from arole in actin filament or myofibril organization.

Attempted division as a response to heart disease

The reactivation of the cardiac cell cycle following myocardialinjury is still a highly controversial topic, with severallaboratories having reported quite divergent rates (for a


review, see [11]). These discrepancies can be explainedpartially by the stringency of cardiomyocyte identificationbut also by the area of the heart that was analyzed since atriashow relatively high rates [55]. While several labs were unableto detect DNA synthesis in e.g. isoproterenol induced hyper-trophic hearts [35], an upregulation of DNA synthesis in theinjured or hypertrophic hearts was reported by others [18] andalso an increase in the activities of cyclin-Cdk complexes wasdescribed during development of pressure overload-inducedLVH in rats [34]. Similarly, it was found that ventricular failureoccurring after acute myocardial infarction [56] or conditionsof global ischemia [57] upregulate the mRNA levels of PCNAand histone-H3. In humans, it was reported repeatedly thatthe ploidy level and number of nuclei per myocyte increasesafter myocardial injury [40,41]. However, none of these studiesdocumented an increase in the number of cardiomyocytessubsequent to hypertrophic stimulation. Thus, it seems thatthe heart is endowed with a program that protects againstuncontrolled proliferation of contracting cardiomyocytes.Only few transgenic models have been shown to overcomethis block at least partially (reviewed in [58]). For example,mice transgenic for cyclin D1 display sustained DNA synthesisin cardiomyocytes at postnatal stages; however, again noincreased division rate was seen and the animals presentedwith a higher rate of multinucleated cardiomyocytes com-pared to control [59]. The only case of dividing adult cardio-myocytes that has been documented so far was by Engel andcolleagues, who reported the division of cultured adult ratcardiomyocytes following p38 MAP kinase inhibition alongwith growth factor stimulation [60]. However, with a mitoticindex of 0.14% this is a rare event even under these specialconditions [60].

Why is division no longer possible?

Why is true cytokinesis not observed in the adult heart in situ(for a review, see [10,52,61])? To complete division, differen-tiated embryonic cardiomyocytes have to disassemble theirmyofibrils completely [3]. This disassembly occurs in twosteps with Z-disk and thin (actin)-filament-associated pro-teins getting disassembled before disassembly of the M-bandsand the thick (myosin) filaments happens [3], a course ofevents that is similar to sarcomere disassembly duringmusclewasting [62]. In muscle wasting the first, potentially rate-limiting step involves the activation of ubiquitous calciumregulated calpains, which degrade titin and α-actinin at theZ-disk and release actin and myosin for ubiquitination andsubsequent degradation by the proteasome [62,63]. We havepreviously suggested a possible role of ubiquitin-dependentdegradation also during myofibril disassembly in dividingcardiomyocytes since this process can be arrested by MG132,a proteasome inhibitor [3]. In the present work, we provideevidence for an involvement of calpain-dependent digestionsince also inhibition of calpain activity halts the disassemblyof myofibrils. This suggests that there may be just one way ofdisassembling such complex structures as myofibrils, whichis conserved in dividing cardiomyocytes as well as in musclewasting and that sequential activation of calpains andsubsequently of developmental stage- and tissue-specific E3ubiquitin ligases is required for this process. Already neonatal

rat cardiomyocytes in culture respond to hypertrophic stimuliin vitro only with multinucleation and no division could beobserved (see Fig. 6). Our hypothesis is that the presence ofstable, highly ordered and functional myofibrils together withan inactivation and downregulation of proteins involved indegradation pathways may be responsible for this inability toundergo full division. During progressive differentiation fromthe embryonic to a postnatal stage a gradual increase in thesize, number and complexity of organization of myofibrils inventricular cardiomyocytes is observed [64,65]. In contrast,atrial cardiomyocytes are smaller in size, about 40% poorer inmyofibrils and appear less differentiated. Interestingly, theyalso retain a higher ability to regenerate both in vivo and invitro [66]. Atrial tumors develop in transgenic mice expressinga fusion of atrial natriuretic factor and SV-40 T-antigen [67],from which dividing differentiated cardiomyocytes can beisolated. Recently, it was also demonstrated that cardiomyo-cyte cell cycle activation in the atrium can antagonize fibrosisand could have a beneficial impact on diseased hearts [68].The less differentiated and simpler cytoarchitecture of atrialcardiomyocytes might also explain why binucleation is lesscommon [64].

In addition to a complex cytoarchitecture, the absence ofdegradation mechanisms, which could disassemble thestable myofibrils in the adult ventricular cardiomyocytes,may prevent division. Our results suggest that calpainactivity is essential for myofibril disassembly and we havepreviously shown that also proteasome activity is required[3]. In addition, we could demonstrate that calpain-1expression levels do not seem to change dramatically duringhypertrophy and that the ubiquitin ligase cullin-3 is notactive in the hypertrophic heart, as judged by the absence ofneddylation. Defects in the ubiquitin–proteasome systemduring cardiomyopathies and in end stage heart failure weresuggested earlier [69,70]. During the progression of compen-sated hypertrophy to heart failure, an activation of ubiqui-tin-dependent degradation mechanisms was proposed [71].It was suggested that ubiquitin is conjugated to contractileor membrane proteins destined for degradation, but that thecomplexes get accumulated due to proteasomal insuffi-ciency, which might eventually cause nuclear fragmentation.The mechanism behind this inhibition of the ubiquitin–proteasome system is not clear but may be related to aphysical plugging of the proteasome core (for a review, see[70]). Downregulation of ubiquitin protein ligases anddysfunction of the proteasome during hypertrophy couldbe another limiting factor that does not allow the disin-tegration of myofibrils to occur in order to facilitate divisionin the adult heart.

In conclusion, our results suggest that cardiomyocytesdownregulate many regulatory and cytoskeletal componentsinvolved in cytokinesis after birth, which may be responsiblefor the uncoupling of cytokinesis from karyokinesis inpostnatal cardiomyocytes. In addition, we show that adultcardiomyocytes attempt to start a mitotic process underhypertrophic conditions by upregulating cytokinesis regulat-ing proteins. At the same time degradation pathways thatmaybe necessary formyofibril disassembly, which is a requisite forcell division of mammalian cardiomyocytes, are only insuffi-ciently activated. These mechanisms, which prevent division


in adult cardiomyocytes, need to be understood in detail tofacilitate myocardial regeneration.

Acknowledgments

We are grateful to all lab members for numerous discussionsand critical comments. In addition, we would like to thank Dr.Izabela Sumara and Prof Matthias Peter for the kind donationof antibodies. The research was supported by grants from theSwiss National Science Foundation (grant number 31.63486/00), the Swiss Federal Institute of Technology (PhD traininggrant), the Gebert Rüf Foundation, the Schweizer Unterrichts-kommission Grant to the Swiss Cardiovascular Teaching andResearch Network and the Swiss Foundation on Research ofMuscle Diseases to JCP.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at doi:10.1016/j.yexcr.2007.01.009.

R E F E R E N C E S

[1] F.J. Manasek, Mitosis in developing cardiacmuscle, J. Cell Biol.37 (1968) 191–196.

[2] A. Hirschy, F. Schatzmann, E. Ehler, J.C. Perriard,Establishment of cardiac cytoarchitecture in the developingmouse heart, Dev. Biol. 289 (2006) 430–441.

[3] P. Ahuja, E. Perriard, J.C. Perriard, E. Ehler, Sequentialmyofibrillar breakdown accompanies mitotic division ofmammalian cardiomyocytes, J. Cell Sci. 117 (2004) 3295–3306.

[4] S. Oparil, S.P. Bishop, F.J. Clubb Jr., Myocardial cellhypertrophy or hyperplasia, Hypertension 6 (1984) III38–III43.

[5] F. Li, X. Wang, J.M. Capasso, A.M. Gerdes, Rapid transition ofcardiac myocytes from hyperplasia to hypertrophy duringpostnatal development, J. Mol. Cell. Cardiol. 28 (1996)1737–1746.

[6] M. Bettencourt-Dias, S. Mittnacht, J.P. Brockes, Heterogeneousproliferative potential in regenerative adult newtcardiomyocytes, J. Cell Sci. 116 (2003) 4001–4009.

[7] K.D. Poss, L.G. Wilson, M.T. Keating, Heart regeneration inzebrafish, Science 298 (2002) 2188–2190.

[8] W.R. MacLellan, M.D. Schneider, Genetic dissection of cardiacgrowth control pathways, Annu. Rev. Physiol. 62 (2000)289–319.

[9] B. Swynghedauw, Are adult cardiocytes still able toproliferate? Arch. Mal. Coeur Vaiss 96 (2003) 1225–1230.

[10] R. von Harsdorf, P.A. Poole-Wilson, R. Dietz, Regenerativecapacity of the myocardium: implications for treatment ofheart failure, Lancet 363 (2004) 1306–1313.

[11] M.H. Soonpaa, L.J. Field, Survey of studies examiningmammalian cardiomyocyte DNA synthesis, Circ. Res. 83(1998) 15–26.

[12] A.P. Beltrami, L. Barlucchi, D. Torella, M. Baker, F. Limana, S.Chimenti, H. Kasahara, M. Rota, E. Musso, K. Urbanek, A. Leri,J. Kajstura, B. Nadal-Ginard, P. Anversa, Adult cardiac stemcells are multipotent and support myocardial regeneration,Cell 114 (2003) 763–776.

[13] H. Oh, S.B. Bradfute, T.D. Gallardo, T. Nakamura, V. Gaussin,Y. Mishina, J. Pocius, L.H. Michael, R.R. Behringer, D.J. Garry,M.L. Entman, M.D. Schneider, Cardiac progenitor cells from

adult myocardium: homing, differentiation, and fusion afterinfarction, Proc. Natl. Acad. Sci. U. S. A. 100 (2003)12313–12318.

[14] K.L. Laugwitz, A. Moretti, J. Lam, P. Gruber, Y. Chen, S.Woodard, L.Z. Lin, C.L. Cai, M.M. Lu, M. Reth, O. Platoshyn, J.X.Yuan, S. Evans, K.R. Chien, Postnatal isl1+ cardioblasts enterfully differentiated cardiomyocyte lineages, Nature 433 (2005)647–653.

[15] M.A. Laflamme, C.E. Murry, Regenerating the heart, Nat.Biotechnol. 23 (2005) 845–856.

[16] E.N. Olson, M.D. Schneider, Sizing up the heart: developmentredux in disease, Genes Dev. 17 (2003) 1937–1956.

[17] V.J. Ferrans, E.R. Rodriguez, Evidence of myocyte hyperplasiain hypertrophic cardiomyopathy and other disorders withmyocardial hypertrophy? Z. Kardiol. 76 (Suppl. 3) (1987) 20–25.

[18] J.M. Capasso, S. Bruno, W. Cheng, P. Li, R. Rodgers, Z.Darzynkiewicz, P. Anversa, Ventricular loading is coupledwith DNA synthesis in adult cardiacmyocytes after acute andchronic myocardial infarction in rats, Circ. Res. 71 (1992)1379–1389.

[19] J.S. Hyams, Cytokinesis: the great divide, Trends Cell Biol. 15(2005) 1.

[20] M. Glotzer, The molecular requirements for cytokinesis,Science 307 (2005) 1735–1739.

[21] M. Glotzer, Animal cell cytokinesis, Annu. Rev. Cell Dev. Biol.17 (2001) 351–386.

[22] A.J. Ridley, Rho-related proteins: actin cytoskeleton and cellcycle, Curr. Opin. Genet. Dev. 5 (1995) 24–30.

[23] Z. Zhao, S.A. Rivkees, Rho-associated kinases play anessential role in cardiac morphogenesis and cardiomyocyteproliferation, Dev. Dyn. 226 (2003) 24–32.

[24] J. Ren, C.X. Fang, Small guanine nucleotide-binding proteinRho and myocardial function, Acta Pharmacol. Sin. 26 (2005)279–285.

[25] F. Matsumura, Regulation of myosin II during cytokinesis inhigher eukaryotes, Trends Cell Biol. 15 (2005) 371–377.

[26] D.N. Drechsel, A.A. Hyman, A. Hall, M. Glotzer, A requirementfor Rho and Cdc42 during cytokinesis in Xenopus embryos,Curr. Biol. 7 (1997) 12–23.

[27] K. Riento, A.J. Ridley, Rocks: multifunctional kinases in cellbehaviour, Nat. Rev., Mol. Cell Biol. 4 (2003) 446–456.

[28] S. Komatsu, T. Yano, M. Shibata, R.A. Tuft, M. Ikebe, Effects ofthe regulatory light chain phosphorylation of myosin II onmitosis and cytokinesis ofmammalian cells, J. Biol. Chem. 275(2000) 34512–34520.

[29] P. Ahuja, Probing the role of septins in cardiomyocytes, Exp.Cell Res. 312 (2006) 1598–1609.

[30] P. Wiesel, L. Mazzolai, J. Nussberger, T. Pedrazzini,Two-kidney, one clip and one-kidney, one clip hypertensionin mice, Hypertension 29 (1997) 1025–1030.

[31] L. Mazzolai, J. Nussberger, J.F. Aubert, D.B. Brunner, G.Gabbiani, H.R. Brunner, T. Pedrazzini, Bloodpressure-independent cardiac hypertrophy induced bylocally activated renin-angiotensin system, Hypertension 31(1998) 1324–1330.

[32] S. Satoh, Y. Ueda, M. Koyanagi, T. Kadokami, M. Sugano, Y.Yoshikawa, N. Makino, Chronic inhibition of Rho kinaseblunts the process of left ventricular hypertrophy leading tocardiac contractile dysfunction in hypertension-inducedheart failure, J. Mol. Cell. Cardiol. 35 (2003) 59–70.

[33] D.A. Guertin, S. Trautmann, D. McCollum, Cytokinesis ineukaryotes, Microbiol. Mol. Biol. Rev. 66 (2002) 155–178.

[34] J.M. Li, R.A. Poolman, G. Brooks, Role of G1 phase cyclins andcyclin-dependent kinases during cardiomyocytehypertrophic growth in rats, Am. J. Physiol. 275 (1998)H814–H822.

[35] M.H. Soonpaa, L.J. Field, Assessment of cardiomyocyte DNAsynthesis during hypertrophy in adult mice, Am. J. Physiol.266 (1994) H1439–H1445.

http://dx.doi.org/doi:10.1016/j.yexcr.2007.01.009


[36] R.K. Kudej, M. Iwase, M. Uechi, D.E. Vatner, N. Oka, Y.Ishikawa, R.P. Shannon, S.P. Bishop, S.F. Vatner, Effects ofchronic beta-adrenergic receptor stimulation in mice, J. Mol.Cell. Cardiol. 29 (1997) 2735–2746.

[37] M. Kuhn, R. Holtwick, H.A. Baba, J.C. Perriard, W. Schmitz, E.Ehler, Progressive cardiac hypertrophy and dysfunction inatrial natriuretic peptide receptor (GC-A) deficientmice, Heart87 (2002) 368–374.

[38] K. Kuwahara, Y. Saito, O. Nakagawa, I. Kishimoto, M. Harada,E. Ogawa, Y. Miyamoto, I. Hamanaka, N. Kajiyama, N.Takahashi, T. Izumi, R. Kawakami, N. Tamura, Y. Ogawa, K.Nakao, The effects of the selective ROCK inhibitor, Y27632, onET-1-induced hypertrophic response in neonatal rat cardiacmyocytes-possible involvement of Rho/ROCK pathway incardiac muscle cell hypertrophy, FEBS Lett. 452 (1999)314–318.

[39] M. Inoko, Y. Kihara, I. Morii, H. Fujiwara, S. Sasayama,Transition from compensatory hypertrophy to dilated, failingleft ventricles in Dahl salt-sensitive rats, Am. J. Physiol. 267(1994) H2471–H2482.

[40] C.A. Beltrami, C. Di Loreto, N. Finato, S.M. Yan, DNA content inend-stage heart failure, Adv. Clin. Path. 1 (1997) 59–73.

[41] G.W. Herget, M. Neuburger, R. Plagwitz, C.P. Adler, DNAcontent, ploidy level and number of nuclei in the humanheart after myocardial infarction, Cardiovasc. Res. 36 (1997)45–51.

[42] X.F. Deng, D.G. Rokosh, P.C. Simpson, Autonomous andgrowth factor-induced hypertrophy in cultured neonatalmouse cardiac myocytes. Comparison with rat, Circ. Res. 87(2000) 781–788.

[43] P.O. Hasselgren, Catabolic response to stress and injury:implications for regulation, World J. Surg. 24 (2000) 1452–1459.

[44] H. Ikeda, T. Oda, K. Kuwano, H. Nakayama, T. Ueno, Y. Koga,H. Toshima, A protease inhibitor, NCO-700, improves thecontractile function in stunned canine myocardium, Jpn.Circ. J. 58 (1994) 713–719.

[45] J. Pines, C. Lindon, Proteolysis: anytime, any place, anywhere?Nat. Cell Biol. 7 (2005) 731–735.

[46] M.D. Petroski, R.J. Deshaies, Function and regulation of cullin-RING ubiquitin ligases, Nat. Rev., Mol. Cell Biol. 6 (2005) 9–20.

[47] T. Kawakami, T. Chiba, T. Suzuki, K. Iwai, K. Yamanaka, N.Minato, H. Suzuki, N. Shimbara, Y. Hidaka, F. Osaka, M.Omata, K. Tanaka, NEDD8 recruits E2-ubiquitin to SCF E3ligase, EMBO J. 20 (2001) 4003–4012.

[48] F. Li, X. Wang, P.C. Bunger, A.M. Gerdes, Formation ofbinucleated cardiac myocytes in rat heart: I. Role ofactin-myosin contractile ring, J. Mol. Cell. Cardiol. 29 (1997)1541–1551.

[49] F.B. Engel, M. Schebesta, M.T. Keating, Anillin localizationdefect in cardiomyocyte binucleation, J. Mol. Cell. Cardiol. 41(2006) 601–612.

[50] S.P. Georgescu, I. Komuro, Y. Hiroi, T. Mizuno, S. Kudoh, T.Yamazaki, Y. Yazaki, Downregulation of polo-like kinasecorrelates with loss of proliferative ability of cardiacmyocytes, J. Mol. Cell. Cardiol. 29 (1997) 929–937.

[51] R.A. Poolman, G. Brooks, Expressions and activities of cellcycle regulatory molecules during the transition frommyocyte hyperplasia to hypertrophy, J. Mol. Cell. Cardiol. 30(1998) 2121–2135.

[52] K.B. Pasumarthi, L.J. Field, Cardiomyocyte cell cycleregulation, Circ. Res. 90 (2002) 1044–1054.

[53] S. Narumiya, S. Yasuda, Rho GTPases in animal cell mitosis,Curr. Opin. Cell Biol. 18 (2006) 199–205.

[54] J.H. Brown, D.P. Del Re, M.A. Sussman, The Rac and Rho hall of

fame: a decade of hypertrophic signaling hits, Circ. Res. 98(2006) 730–742.

[55] P.P. Rumyantsev, A. Borisov, DNA synthesis inmyocytes fromdifferent myocardial compartments of young rats in norm,after experimental infarction and in vitro, Biomed. Biochim.Acta 46 (1987) S610–S615.

[56] K. Reiss, W. Cheng, A. Giorando, A. De Luca, B. Li, J. Kajstura, P.Anversa, Myocardial infarction is coupled with activation ofcyclins and cyclin-dependent kinases in myocytes, Exp. CellRes. 225 (1996) 44–54.

[57] K. Reiss, J. Kajstura, J.M. Capasso, T.A. Marino, P. Anversa,Impairment of myocyte contractility following coronaryartery narrowing is associated with activation of the myocyteIGF1 autocrine system, enhanced expression of late growthrelated genes, DNA synthesis, and myocyte nuclear mitoticdivision in rats, Exp. Cell Res. 207 (1993) 348–360.

[58] L.J. Field, Modulation of the cardiomyocyte cell cycle ingenetically altered animals, Ann. N. Y. Acad. Sci. 1015 (2004)160–170.

[59] M.H. Soonpaa, G.Y. Koh, L. Pajak, S. Jing, H. Wang, M.T.Franklin, K.K. Kim, L.J. Field, Cyclin D1 overexpressionpromotes cardiomyocyte DNA synthesis andmultinucleationin transgenic mice, J. Clin. Invest. 99 (1997) 2644–2654.

[60] F.B. Engel, M. Schebesta, M.T. Duong, G. Lu, S. Ren, J.B.Madwed, H. Jiang, Y. Wang, M.T. Keating, p38 MAP kinaseinhibition enables proliferation of adult mammaliancardiomyocytes, Genes Dev. 19 (2005) 1175–1187.

[61] F.B. Engel, Cardiomyocyte proliferation: a platform formammalian cardiac repair, Cell Cycle 4 (2005) 1360–1363.

[62] M.C. Lebart, Y. Benyamin, Calpain involvement in theremodeling of cytoskeletal anchorage complexes, FEBS J. 273(2006) 3415–3426.

[63] P.O. Hasselgren, M.J. Menconi, M.U. Fareed, H. Yang, W. Wei,A. Evenson, Novel aspects on the regulation of musclewasting in sepsis, Int. J. Biochem. Cell Biol. 37 (2005)2156–2168.

[64] P.P. Rumyantsev, Interrelations of the proliferation anddifferentiation processes during cardiac myogenesis andregeneration, Int. Rev. Cytol. 51 (1977) 186–273.

[65] J.C. Perriard, A. Hirschy, E. Ehler, Dilated cardiomyopathy. Adisease of the intercalated disc? Trends Cardiovasc. Med. 13(2003) 30–38.

[66] P.P. Rumyantsev, V.O. Marakjan, Reactive synthesis of DNAand mitotic division in atrial heart muscle cells followingventricle infarction, Experientia 24 (1968) 1234–1235.

[67] M.E. Steinhelper, N.A. Lanson Jr., K.P. Dresdner, J.B.Delcarpio, A.L. Wit, W.C. Claycomb, L.J. Field, Proliferation invivo and in culture of differentiated adult atrialcardiomyocytes from transgenic mice, Am. J. Physiol. 259(1990) H1826–H1834.

[68] H. Nakajima, H.O. Nakajima, K. Dembowsky, K.B. Pasumarthi,L.J. Field, Cardiomyocyte cell cycle activation amelioratesfibrosis in the atrium, Circ. Res. 98 (2006) 141–148.

[69] J. Weekes, K. Morrison, A. Mullen, R. Wait, P. Barton, M.J.Dunn, Hyperubiquitination of proteins in dilatedcardiomyopathy, Proteomics 3 (2003) 208–216.

[70] S.R. Powell, The ubiquitin proteasome system in cardiacphysiology and pathology, Am. J. Physiol.: Heart Circ. Physiol.291 (2006) H1–H19.

[71] S. Hein, E. Arnon, S. Kostin, M. Schonburg, A. Elsasser, V.Polyakova, E.P. Bauer, W.P. Klovekorn, J. Schaper, Progressionfrom compensated hypertrophy to failure in the pressure-overloaded human heart: structural deterioration andcompensatory mechanisms, Circulation 107 (2003) 984–991.

re-expression of proteins involved in cytokinesis during cardiac hypertrophy

Documents