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A Cyclin D2-Rb Pathway Regulates Cardiac Myocyte Size and RNA Polymerase III After Biomechanical Stress in Adult Myocardium

A Cyclin D2-Rb Pathway Regulates Cardiac Myocyte Size and RNA Polymerase III After Biomechanical Stress in Adult Myocardium
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  A Cyclin D2–Rb Pathway Regulates Cardiac Myocyte Sizeand RNA Polymerase III After Biomechanical Stress inAdult Myocardium Ekaterini Angelis, Alejandro Garcia, Shing S. Chan, Katja Schenke-Layland, Shuxen Ren,Sarah J. Goodfellow, Maria C. Jordan, Kenneth P. Roos, Robert J. White, W. Robb MacLellan  Abstract —Normally, cell cycle progression is tightly coupled to the accumulation of cell mass; however, the mechanismswhereby proliferation and cell growth are linked are poorly understood. We have identified cyclin (Cyc)D2, a G 1  cyclinimplicated in mediating S phase entry, as a potential regulator of hypertrophic growth in adult post mitotic myocardium.To examine the role of CycD2 and its downstream targets, we subjected CycD2-null mice to mechanical stress.Hypertrophic growth in response to transverse aortic constriction was attenuated in CycD2-null compared withwild-type mice. Blocking the increase in CycD2 in response to hypertrophic agonists prevented phosphorylation of CycD2-target Rb (retinoblastoma gene product) in vitro, and mice deficient for Rb had potentiated hypertrophic growth.Hypertrophic growth requires new protein synthesis and transcription of tRNA genes by RNA polymerase (pol) III,which increases with hypertrophic signals. This load-induced increase in RNA pol III activity is augmented inRb-deficient hearts. Rb binds and represses Brf-1 and TATA box binding protein (TBP), subunits of RNA polIII–specific transcription factor B, in adult myocardium under basal conditions. However, this association is disruptedin response to transverse aortic constriction. RNA pol III activity is unchanged in CycD2   /   myocardium after transverseaortic constriction, and there is no dissociation of TBP from Rb. These investigations identify an essential role for theCycD2-Rb pathway as a governor of cardiac myocyte enlargement in response to biomechanical stress and, morefundamentally, as a regulator of the load-induced activation of RNA pol III.  ( Circ Res . 2008;102:1222-1229.)Key Words:  hypertrophy    genetically altered mice    animal models of human disease   cell signaling/signal transduction D uring development, cell cycle progression is closelycoupled to the accumulation of cell mass to maintain auniform cell size. It has been suggested that proteins involvedin cell cycle progression control both processes, therebylinking proliferation and cell growth. 1 In the fetal heart, theproliferative activity of cardiac myocytes is accompanied byincreases in cell mass, thereby maintaining myocyte size.However, in adult cardiac myocytes cell growth becomesuncoupled from proliferation after growth stimuli, resulting inhypertrophic growth. 2 The molecular mechanisms by whichproliferation and cell growth are coupled in myocytes arepoorly understood; however, cell cycle regulatory proteinstypically associated with G 1  exit have also been implicated inregulating cardiac myocyte cell growth. 3–5 Cyclin (Cyc)D-Cdk4 in particular has been associated with cardiac hypertro-phy, although the downstream effectors have not been iden-tified. 6,7 Previous studies have indicated that inhibiting G 1 cyclin/Cdk activity in adult, postmitotic, cardiac myocytescan attenuate hypertrophic growth. 8,9 Consistent with thisfinding, we recently reported that Myc-induced hypertrophicgrowth was dependent on the presence of CycD2, similar towhat has been reported for Myc-induced cell cycle reentrybut was independent of Cdk2 activity. 10 The association between CycD and Cdk4 represents therate-limiting step in a complex cascade that leads to the phos-phorylation of a number of proteins, including the retinoblas-toma gene product, or Rb. 8,11–13 When phosphorylated Rbbecomes inactive, its association with a family of transcriptionfactors known as E2Fs is disrupted, thereby allowing them toactivate transcription. 14 Previous studies have determined thatRb phosphorylation by CycD-Cdk4 is essential for cell cycleprogression, largely by regulating the transcriptional activities of these E2Fs. 15,16 However, whether this signaling pathway is alsoimportant in cardiac hypertrophy is unknown. Among the Original received September 6, 2007; revision received March 18, 2008; accepted April 4, 2008.From the Cardiovascular Research Laboratory, Departments of Medicine (E.A., A.G., S.S.C., K.S.-L., W.R.M.), Physiology (M.C.J., K.P.R., W.R.M.),and Anesthesiology (S.R.), David Geffen School of Medicine at the University of California, Los Angeles; and Institute of Biomedical and Life Sciences(S.J.G., R.J.W.), University of Glasgow, United Kingdom.This manuscript was sent to Stephen F. Vatner, Consulting Editor, for review by expert referees, editorial decision, and final disposition.Correspondence to W. Robb MacLellan, The Cardiovascular Research Laboratory, David Geffen School of Medicine at UCLA, 675 C E Young Dr,MRL 3-645, Los Angeles, CA 90095-1760. E-mail© 2008 American Heart Association, Inc. Circulation Research  is available at DOI: 10.1161/CIRCRESAHA.107.163550 1222 Cellular Biology  by guest on December 29, 2015 Downloaded from by guest on December 29, 2015 Downloaded from by guest on December 29, 2015 Downloaded from by guest on December 29, 2015 Downloaded from by guest on December 29, 2015 Downloaded from by guest on December 29, 2015 Downloaded from by guest on December 29, 2015 Downloaded from by guest on December 29, 2015 Downloaded from by guest on December 29, 2015 Downloaded from by guest on December 29, 2015 Downloaded from by guest on December 29, 2015 Downloaded from   known hypertrophic signaling cascades, a direct mechanisticlink to Rb is perhaps best suggested for its interaction andregulation of the RNA polymerases. 17,18 Therefore, to understand the role of CycD2 and Rb proteinsduring cardiac hypertrophy, and to determine the mechanismby which these cell cycle regulators modulate cardiac hyper-trophy in vivo, we used mouse models deficient in theseproteins. Mice in which CycD2 has been genetically ablatedare viable and display normal cardiac anatomy and function. 19 Although normal at baseline, CycD2-deficient mice exhibitedattenuated hypertrophy in response to pressure overload. Todetermine the importance of Rb as a downstream target, weused mice with a cardiac-specific deletion of Rb. These micehave normal heart size at baseline, 20 but hypertrophic growthin response to mechanical or pharmacological stress wasincreased significantly in Rb-null mice. Furthermore, activityof RNA polymerase (pol) III was increased in Rb-deficienthearts subjected to hemodynamic stress, whereas this trans-verse aortic constriction (TAC)-induced increased activitywas not seen in CycD2   /   myocardium. Thus, we proposethat CycD and Rb work in an interdependent manner tocontrol myocyte size in postmitotic myocardium after me-chanical stress. Our data identify CycD2, through its phos-phorylation target Rb, as a pivotal regulator of RNA pol IIIactivation in myocardial hypertrophy. Materials and Methods Animal Studies Cardiac-restricted Rb-deficient mice (CRb L/L ) have been described. 20 CycD2-deficient mice were a kind gift from Dr P. Sicinski. 19 ForTAC, a fixed pressure overload was obtained by surgically constrict-ing the transverse aorta, as described. 21 Genotypes of mice weredetermined by PCR as described. 10,20 All animals were handled andmaintained in accordance with institutional guidelines and the NIHGuide for the Care and Use of Laboratory Animals. Isolation of Cardiac Myocytes and Analysis Neonatal rat ventricular myocytes (NRVMs) were prepared asdescribed. 10,20 Myocyte dimensions were determined and volumeswere calculated using a computerized morphometric system. 21 De-tails for viral propagation 22 are available in the online data supple-ment at For small interfering (si)RNAstudies, NRVMs were transfected with 125 nmol/L CycD2 siRNA ornonspecific siRNA (Qiagen) with Lipofectamine 2000 (Invitrogen)according to the specifications of the manufacturer. Histology and Immunostaining For histology, hearts were either frozen in OCT compound or fixedovernight in 4% paraformaldehyde buffered with PBS and routinelyprocessed. Details of immunostaining protocols and antibodies areavailable in the online data supplement. RNA and Protein Analysis Total RNA was extracted (Tri Reagent, Sigma) according to theinstructions of the manufacturer. Northern blot and ribonucleicprotection assays were conducted as previously described. 20,23 Real-time quantitative PCR was conducted using the ABI PRISM 7700Sequence Detection System; TaqMan (Applied Biosystems, FosterCity, Calif). Primer sequences for all genes analyzed in the presentstudy have been previously reported or are available on request. 23,24 Western blotting and immunoprecipitations were performed as wehave described, 10,25 and details of the antibodies used are available inthe online data supplement. RNA Pol III Assays RT-PCR analysis of ARPP P0 mRNA and primary tRNA Tyr ortRNA Leu transcripts was carried out as previously described. 26,27 Gelswere scanned and quantitated by a UVP image analysis system(Adobe PHOTOSHOP 4.0, Adobe Systems, Mountain View, Calif). Statistical Analysis All data are presented as means  SEM. Results were compared by1-way ANOVA and Fisher’s protected least significant difference orTukey’s multiple comparison post tests, using significance at aprobability value of   0.05. Results CycD2-Null Mice Display Attenuated Hypertrophyin Response to Pressure Overload Our previous work had shown that the hypertrophic effects of Myc were mediated through a CycD2-dependent pathway. 10 Inaccordance with previous studies, 4 CycD2 protein expressionincreases both in vitro and in vivo in response to hypertrophicstimuli (Figure I in the online data supplement). To determinewhether the importance of CycD2 was specific to Myc-inducedhypertrophy or whether it was more generally important inregulating hypertrophic growth, we subjected CycD2-null miceto pressure overload for 14 days. Homozygous CycD2-deficientmice are viable and appear normal but are sterile. 19 Although notdescribed in the srcinal report, CycD2   /   mice had a 22.5%reduction in body weight compared with wild-type littermates at12 weeks (supplemental Table I; 23.5  1.6 g [CycD2   /   ] versus28.8  0.9 g [CycD2   /   ];  P  0.05); however, the heart-to-bodyweight ratios (HW/BW) were similar at baseline (4.35  0.14mg/g [CycD2   /   ] versus 4.40  0.16 mg/g [CycD2   /   ];  P  NS).As expected, TAC increased CycD2 mRNA and protein levelsin wild-type but not CycD2-deficient mice (Figure 1A and 1B).CycD2 mRNA increased 75% in CycD2   /   mice after TAC, asmeasured by real-time PCR analyses (1.00  0.18 versus1.75  0.19;  P  0.05). Similarly, CycD2 protein expression in-creased 73% in CycD2   /   mice after TAC as measured byWesternblotting(1.62  0.36versus2.80  0.40; P  0.05).Therewasa64%reductioninthehypertrophicresponseinCycD2-nullmice compared with wild-type mice subjected to TAC( P  0.02). Two weeks of TAC induced a 25% increase in theHW/BW in CycD2 wild-type mice (Figure 1C; HW/BW:4.35  0.14 versus 5.45  0.29 mg/g;  P  0.005). In contrast,CycD2-null mice displayed an attenuated hypertrophic responsewith only a 9% increase in HW/BW (Figure 1C; HW/BW:4.40  0.16 versus 4.81  0.14 mg/g;  P  0.05). Fetal cardiacgenes including atrial and B-type natriuretic peptides, as well as  -skeletal actin, were upregulated in response to TAC in bothventricles from mice of both genotypes despite the differences inmyocardial mass, suggesting that not all aspects of the hyper-trophic response were impaired (Figure 1D). Rb Regulates Hypertrophic Growth In Vitro Although several CycD2 phosphorylation targets are presentin adult cardiac muscle, Rb is the primary target of CycD-Cdk4 kinase complexes and has been implicated, at leastindirectly, in regulating protein synthesis. 7 Thus, we sought todetermine whether phosphorylation of Rb in response tohypertrophic agonists was dependent on CycD2. To knock down CycD2 expression, we transfected NRVMs with eithernonspecific siRNA or a specific siRNA to CycD2. As shown Angelis et al CycD2-Rb Pathway Regulates Cardiac Hypertrophy  1223  by guest on December 29, 2015 Downloaded from   in Figure 2A, siRNA transfection was able to induce a 98%decrease in CycD2 protein expression in NRVMs comparedwith nonspecific siRNA treatment (1.37  0.29 versus0.03  0.01;  P  0.05). To determine whether depleting CycD2impaired Rb phosphorylation in response to hypertrophicagonists, NRVMs transfected with nonspecific or CycD2siRNA and stimulated with phenylephrine (PE) were immu-nostained with an antibody specific for phosphorylated serine780 on Rb (pRb S780 ), a target of CycD-Cdk4. As expected,treatment of NRVMs with PE resulted in the increasedexpression of both CycD2 and pRb S780 in nonspecific siRNA–transfected cells (Figure 2B, a and b). However, transfectionof NRVMs with CycD2 siRNA blocked the increase inCycD2 seen with PE and diminished Rb S780 phosphorylation(Figure 2B, c and d). To determine the role of Rb inregulating hypertrophic growth, we stimulated NRVMs withPE and measured protein accumulation and myocyte sizewith or without Rb overexpression. Infection of NRVMs withAdRb resulted in high levels of Rb expression (Figure 2C),which had no effect on basal amino acid incorporation inquiescent myocytes (Figure 2C). PE increased relative proteinsynthesis rates by 33.4% in AdLacZ-infected cultures(1.33  0.1 versus 1.0  0.02 for vehicle-treated, LacZ-infected cells;  P  0.01). This increase in protein synthesiswas blocked completely by Rb (0.91  0.07 versus 0.91  0.09for AdRb infected myocytes treated with PE versus vehicle,respectively;  P  NS, Figure 2C and 2D). Pressure Overload Results in Enhanced Hypertrophyin Adult Rb-Deficient Cardiac Myocytes In Vivo To determine the role of Rb in regulating the hypertrophicresponse in adult cardiac myocytes in vivo, we subjectedcardiac-restricted Rb-deficient (CRb L/L ) mice we had previ-ously created to TAC. 20 These mice are phenotypically andbiochemically normal at baseline. Hearts from Rb-deficientCRb L/L mice were significantly larger than those from controlCRb   /   mice after being subjected to 1 week of TAC(supplemental Table II; HW/BW: 6.40  0.34 versus5.25  0.25 mg/g;  P  0.05). Likewise, at 2 weeks (Figure 3A),CRb   /   mice developed a 39.3% increase in HW/BW (HW/ BW: 5.23  0.3 versus 4.21  0.16 mg/g for sham-operatedmice;  P  0.05), whereas Rb-deficient CRb L/L mice demon-strated a 59.4% increase (HW/BW: 6.44  0.5 versus 4.41  0.19mg/g for sham-operated mice;  P  0.001). This enhancement of myocardial mass in CRb L/L mice compared with CRb   /   animalsafter 2 weeks of banding was also significant (Figure 3A; P  0.05). Although the role of Rb in mediating cell cycle exit instriated muscle is controversial, 28 increased myocyte prolifera-tioncannotexplaintheincreaseincardiacmassthatweobservedbecause no evidence of myocyte cell cycle reentry was seeneither by 5-bromodeoxyuridine incorporation (Figure 3B) orKi-67 expression (not shown). In contrast, myocyte fiber widthwas significantly increased in CRb L/L mice subjected to TACwhen compared with sham-operated CRb L/L or CRb   /   mice Figure 1.  CycD2   /   mice display attenuatedhypertrophy in response to pressure overload. A, Representative ribonucleic protection assayand quantification of CycD2 mRNA by real-timePCR normalized to GAPDH (n  4 per group).* P  0.05 CycD2 mRNA in CycD2   /   after TACcompared with CycD2   /   sham. B, Westernblotting on total ventricular lysates after 2weeks of TAC from the indicated genotypesand conditions. Protein quantification revealedan increase in CycD2 protein expression inCycD2   /   hearts after TAC. Cardiac actin(   -CaA) was used to normalize total proteinlevels (n  4 for each group). * P  0.05 forCycD2   /   TAC as compared with sham. C, Twoweeks of TAC induced a significant increase inthe HW/BW (mg/g) in CycD2   /   but notCycD2   /   mice (n  5 per group). *  P  0.005 forCycD2   /   TAC compared with CycD2   /   sham. An increase in the HW/BW in CycD2   /   mice inresponse to TAC was not observed. D, North-ern blot on total ventricular RNA. Expressionsof atrial (ANP) and B-type (BNP) natriureticpeptides and skeletal actin (   -SkA) and 18Sribosomal RNA (18S rRNA) as a loading controlare shown. 1224 Circulation Research  May 23, 2008  by guest on December 29, 2015 Downloaded from   (12.72  1.26 versus 9.61  0.35 or 10.05  0.65   m;  P  0.05;Figure 3C and 3D), suggesting the increased heart size wasrelated to myocyte hypertrophy. This enhanced hypertrophicresponse could not be accounted for by extrinsic or intrinsichemodynamic differences, because the gradient across the con-striction at 2 weeks did not differ significantly between thegenotypes (51.5  9.8 versus 60.7  4.6 mm Hg;  P  NS), andbecause left ventricular function was normal for bothgenotypes by 2D echo or invasive hemodynamics. Mea-surements of contractility (  dP/dt; 5003  862 versus Figure 2.  CycD2 phosphorylates and inacti-vates Rb, which regulates protein synthesis invitro. NRVMs were transfected with either non-specific siRNA (NS siRNA) or specific CycD2siRNA and treated with 100 mmol/L PE for 24hours. A, Western blot analysis reveals a 98%knockdown of CycD2 protein expression inCycD2 siRNA–transfected NRVMs (n  3 percondition;  P  0.05). B, a and b, Immunostainingof NRVMs treated with nonspecific siRNA revealed robust staining of CycD2 andphospho-Rb S780 (pRb S780  ) in response to PE.B, c and d, CycD2 siRNA–transfected cellsrevealed diminished expression of both CycD2and pRb S780 in response to PE. Scalebar  50   m. C and D, NRVMs cultured inserum-free media for 48 hours and infectedwith the indicated virus. NRVM cultures werethen stimulated with 100   mol/L PE for 24hours. C, Relative levels of Rb expressionwere determined by Western blotting. 3 H-phenylalanine incorporation was quantifiedand normalized to unstimulated AdLacZ cul-tures. The pooled results of 3 experiments arepresented. * P  0.01 vs unstimulated AdlacZmyocytes and  P  0.001 vs AdRb-infectedcultures. Figure 3.  Rb-deficient mice display an exag-gerated hypertrophic response in vivo. Control(CRb   /    ) or Rb-deficient (CRb L/L  ) mice under-went sham or TAC surgery and were followedfor 2 weeks (n  6 for each group). A, Heartweights (mg) were normalized to body weight(g). * P  0.05 for CRb   /   TAC vs CRb   /   shamand CRb L/L sham, ** P  0.05 for CRb L/L TAC vsCRb   /   TAC, and  P  0.0001 vs CRb   /   shamand CRb L/L sham. B, Percentage of 5-bromode-oxyuridine–positive (BrdU  ve) nuclei fromsham and TAC mice were quantified (n  6 pergroup;  P  NS). C and D, Fiber width of wheatgerm agglutinin–stained hearts from sham andTAC mice were quantified (n  5 in each group).D, Representative wheat germ agglutinin–stained myocardial sections from CRb   /   (aand b) or CRb L/L (c and d) subjected to sham(a and c) or TAC (b and d) surgery. Angelis et al CycD2-Rb Pathway Regulates Cardiac Hypertrophy  1225  by guest on December 29, 2015 Downloaded from   4243  313 mm Hg/sec;  P  NS) and relaxation (  dP/dt;  6456  1329 versus  5171  592 mm Hg/sec;  P  NS) wereindistinguishable between CRb   /   and CRb L/L mice subjectedto TAC for 2 weeks.To determine whether the exaggerated hypertrophic re-sponse to load in Rb-null myocardium was generalizable toother hypertrophic stimuli, we next subjected mice to aninfusion of isoproterenol (ISO) versus the vehicle, for oneweek. ISO treatment caused a 26.8% increase in HW/BW incontrol CRb   /   mice (HW/BW: 4.63  0.08 versus 5.87  0.24mg/g;  P  0.0001), but Rb-deficient CRb L/L mice demon-strated a 36.4% increase (HW/BW: 4.67  0.11 versus6.37  0.17 mg/g;  P  0.001). This induced HW/BW wassignificantly greater in ISO-treated CRb L/L animals than inISO-stimulated CRb   /   mice (6.37  0.17 versus 5.87  0.24mg/g;  P  0.05). This increase in cardiac mass paralleled acorresponding increase in individual myocyte volume in theRb-deleted background. Isolated Rb-deficient myocytes fromISO-treated CRb L/L ventricles demonstrated a 25.1% increasedin myocyte volume when compared with similarly treatedCRb   /   myocytes (21962  1247 versus 17549  1891   m 3 ; P  0.09). Thus, in adult myocardium, Rb primarily functionsto constrain myocyte size in response to biomechanical stress. Expression of E2Fs and E2F Target Genes IsUnchanged in Rb-Deficient Myocardium Although Rb associates with many proteins, classically itbinds and inhibits members of the E2F family of transcriptionfactors. Because E2Fs have been implicated in regulatingcardiac hypertrophy, at least in vitro, 29 we examined theexpression of E2F family members in CRb   /   or CRb L/L miceat baseline and after TAC with real-time PCR. E2F-1 mRNAincreased 3-fold in CRb   /   hearts in response to TACcompared with baseline (Figure 4A; 1.00  0.08 versus3.05  0.83;  P  0.05); however, there were no differences inexpression between CRb   /   and CRb L/L mice (Figure 4A).Interestingly, no significant change was noted with TAC inthe expression of E2F-3, as determined by semiquantitativePCR analysis, which was the E2F family member previouslyassociated with hypertrophic growth in cultured NRVMs(Figure 4A). 29 No change in either E2F-4 or -5 expressionwas seen in response to TAC. Because E2F transcriptionalactivity could be altered in the absence of Rb, even if expression levels were not, we examined the levels of a panelof E2F-1–dependent target genes. 24 Semiquantitative PCRrevealed no significant differences in the expression of any of these E2F-target genes in CRb   /   and CRb L/L myocardiumafter TAC (Figure 4B). Thus, differences in E2F expressionor activity are unlikely to account for the enhanced hypertro-phic response in Rb-null myocardium. Pressure Overload Induces Enhanced RNA Pol IIIActivity in Rb-Null Hearts RNA pol III activity is known to increase in cardiac myocytessubjected to hypertrophic signals; however, the mechanismsregulating this effect are poorly understood. 30 Rb is known tonegatively regulate RNA pol III activity, 17 via binding toBrf-1 and TATA box binding protein (TBP), resulting ininhibition of the pol III–specific transcription factor complexTFIIIB. 25,31 To clarify the relationship between Rb and these2 main subunits of TFIIIB, in response to a hypertrophicstimulus, we performed immunoprecipitation studies usingtotal ventricular extracts from wild-type mice exposed toTAC for 7 days. Expression of phosphorylated and total Rb,Brf-1, and TBP in sham and TAC ventricles are displayed inFigure 5A and 5B. Although total expression levels of thesefactors did not change with TAC, phosphorylation of Rbincreased in hypertrophic ventricles (Figure 5B). Brf-1 andTBP associated with Rb in wild-type sham ventricles (Figure5C), but phosphorylation of Rb in banded ventricles disrupted Figure 4.  Expression and activity of E2F familymembers is not altered in Rb-null myocardiumafter TAC. A, Representative E2F-1, -3, -4, and-5 expression obtained from semiquantitativePCR analyses. Quantification via real-time PCRanalysis (bar graph) revealed a 3-fold increasein E2F-1 mRNA expression in response to TACin CRb   /   mice (n  3 to 4 per condition).* P  0.05. No difference was noted in the mRNA expression of E2F-3, -4, and -5. B, Semiquanti-tative PCR analyses of E2F-1 target genes wasexamined in each genotype in response toTAC. TK-1 indicates thymidine kinase-1; TopoII-  , topoisomerase II-  . 1226 Circulation Research  May 23, 2008  by guest on December 29, 2015 Downloaded from 
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